Binding acceleration techniques for the detection of analytes

ABSTRACT

The invention relates to compositions and methods useful in the acceleration of binding of target analytes to capture ligands on surfaces. Detection proceeds through the use of an electron transfer moiety (ETM) that is associated with the target analyte, either directly or indirectly, to allow electronic detection of the ETM.

This application is a continuing application of U.S. Ser. No.09/134,058, filed Aug. 14, 1998 and claims the benefit of U.S.Provisional Application Ser. No. 60/090,389, filed Jun. 23, 1998.

FIELD OF THE INVENTION

The invention relates to compositions and methods useful in theacceleration of binding of target analytes to capture ligands onsurfaces. Detection proceeds through the use of an electron transfermoiety (ETM) that is associated with the target analyte, either directlyor indirectly, to allow electronic detection of the ETM.

BACKGROUND OF THE INVENTION

There are a number of assays and sensors for the detection of thepresence and/or concentration of specific substances in fluids andgases. Many of these rely on specific ligand/antiligand reactions as themechanism of detection. That is, pairs of substances (i.e. the bindingpairs or ligand/antiligands) are known to bind to each other, whilebinding little or not at all to other substances. This has been thefocus of a number of techniques that utilize these binding pairs for thedetection of the complexes. These generally are done by labelling onecomponent of the complex in some way, so as to make the entire complexdetectable, using, for example, radioisotopes, fluorescent and otheroptically active molecules, enzymes, etc.

Other assays rely on electronic signals for detection. Of particularinterest are biosensors. At least two types of biosensors are known;enzyme-based or metabolic biosensors and binding or bioaffinity sensors.See for example U.S. Pat. Nos. 4,713,347; 5,192,507; 4,920,047;3,873,267; and references disclosed therein. While some of these knownsensors use alternating current (AC) techniques, these techniques aregenerally limited to the detection of differences in bulk (ordielectric) impedance.

The use of electrophoresis in microfluidic methods to facilitate thebinding of biological molecules to their binding partners for subsequentdetection is known; see for example U.S. Pat. Nos. 5,605,662 and5,632,957, and references disclosed therein.

Similarly, electronic detection of nucleic acids using electrodes isalso known; see for example U.S. Pat. Nos. 5,591,578; 5,824,473;5,705,348; 5,780,234 and 5,770,369; U.S. Ser. No. 08/911,589; and WO98/20162; PCT/US98/12430; PCT/US98/12082; PCT/US99/10104;PCT/US99/01705, and PCT/US99/01703.

One of the significant hurdles in biosensor applications is the rate atwhich the target analyte binds to the surface for detection and theaffinity for the surface. There are a number of techniques that havebeen developed in nucleic acid applications to either accelerate therate of binding, or to concentrate the sample at the detection surface.These include precipitation of nucleic acids (see EP 0 229 442 A1,including the addition of detergents (see Pontius et al., PNAS USA88:8237 (1991)); partitioning of nucleic acids in liquid two phasesystems (see Albertsson et al., Biochimica et Biophysica Acta 103:1-12(1965), Kohne et al., Biochem. 16(24):5329 (1977), and Müller,Partitioning of Nucleic Acids, Ch. 7 in Partitioning in AqueousTwo-Phase Systems, Academic Press, 1985)), as well as partitioning inthe presence of macroligands (see Müller et al., Anal. Biochem. 118:269(1981)); and the addition of nucleic acid binding proteins (see Pontiuset al., PNAS USA 87:8403 (1990) and U.S. Pat. No. 5,015,569), all ofwhich are expressly incorporated by reference. In addition, partitioningsystems for some proteins have also been developed, see Gineitis et al.,Anal. Biochem. 139:400 (1984), also incorporated by reference.

However, there is a need for a system that combines acceleration ofbinding of target analytes, including nucleic acids, to a detectionelectrode for subsequent electronic detection.

SUMMARY OF THE INVENTION

In accordance with the objects outlined above, the present inventionprovides methods of detecting a target analyte in a sample. The methodscomprise concentrating the target analyte in a detection chambercomprising a detection electrode comprising a covalently attachedcapture ligand. The target analyte is bound to the capture ligand toform an assay complex comprising at least one electron transfer moiety(ETM). The presence of the ETM is then detected using the detectionelectrode.

In a further aspect, the concentration step comprises placing the samplein an electric field between at least a first electrode and at least asecond electrode sufficient to cause electrophoretic transport of thesample to the detection electrode.

In an additional aspect, the concentration step comprises including atleast one volume exclusion agent in the detection chamber.

In a further aspect, the concentration step comprises comprisesprecipitating the target analyte.

In an additional aspect, the concentration step comprises including atleast two reagents that form two separable solution phases, such thatthe target analyte concentrates in one of the phases.

In a further aspect, the concentration step comprises binding the targetanalyte to a shuttle particle.

In an additional aspect, the invention provides methods of detectingtarget analytes comprising flowing the sample past a detection electrodecomprising a covalently attached capture ligand under conditions thatresult in the formation of an assay complex. As above, the assay complexfurther comprises at least one electron transfer moiety (ETM), and thepresence of the ETM is detected using said detection electrode.

In a further aspect, the methods are for the detection of target nucleicacids and include the use of hybridization accelerators. The assaycomplex is formed in the presence of a hybridization accelerator, thatmay be a nucleic acid binding protein or a polyvalent ion.

In an additional aspect, the invention provides methods of detecting atarget analyte in a sample comprising adding the sample to a detectionelectrode comprising a covalently attached capture ligand underconditions that result in the formation of an assay complex. Theconditions include the presence of mixing particles.

In a further aspect, the invention provides substrates comprising aplurality of gold electrodes. Each gold electrode comprises aself-assembled monolayer, a capture ligand, and an interconnect suchthat each electrode is independently addressable. Preferred substratesinclude printed circuit board materials such as fiberglass.

In an additional aspect, the invention provides methods of making asubstrate comprising a plurality of gold electrodes. The methodscomprise coating an adhesion metal onto a fiberglass substrate, andcoating gold onto the adhesion metal. A pattern is then formed usinglithography, and the pattern comprises the plurality of electrodes andassociated interconnects. The methods optionally include adding aself-assembled monolayer (SAM) to each electrode.

In an additional aspect, the invention provides methods of making asubstrate comprising a plurality of gold electrodes. The methodscomprise coating an adhesion metal onto a substrate, and coating goldonto the adhesion metal. A pattern is then formed using lithography, andthe pattern comprises the plurality of electrodes and associatedinterconnects. The methods further include adding a self-assembledmonolayer (SAM) to each electrode.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C, 1D, 1E and 1F depict several representativeconfigurations of the use of two sets of electrodes, an electrophoresisset and a detection set. In FIGS. 1A and 1B, a substrate 30 has a firstelectrophoresis electrode 10 with detection electrodes 20 either on topor embedded in but electrically isolated from the electrophoresiselectrode. There is a sample receiving area 40 as well. The counterelectrode for the electrophoresis and detection electrodes are notshown. FIG. 1C represents a side view of FIG. 1A, with the addition ofthe counter electrophoresis electrode 50 and optionally the counterdetection electrode 60. A permeation layer 25 is also shown. As will beappreciated by those in the art, these counter electrodes may be thesame electrode, if they are used sequentially. FIGS. 1D, 1E and 1Fdepicts the use of individual electrophoresis electrodes. FIG. 1E is aside view of 1D. FIG. 1F shows the configuration for sequentially movinga sample from one detection electrode to another, as is more fullydescribed below.

FIG. 2 depicts the use of multidimensional arrays of electrophoresiselectrodes for both spatial targeting of the sample as well as “mixing”to increase binding kinetics. FIG. 2 shows electrophoresis electrodes 10and detection electrodes 20. Electrophoresis voltage applied as betweenelectrophoretic electrodes 10 and 15 and, at the same time,electrophoretic electrodes 12 and 17, can drive the target analyte todetector electrode 20.

FIGS. 3A, 3B and 3C depict three preferred embodiments for attaching atarget sequence to the electrode. FIG. 3A depicts a target sequence 120hybridized to a capture probe 100 linked via a attachment linker 106,which as outlined herein may be either a conductive oligomer or aninsulator. The electrode 105 comprises a monolayer of passivation agent107, which can comprise conductive oligomers (herein depicted as 108)and/or insulators (herein depicted as 109). As for all the embodimentsdepicted in the figures, n is an integer of at least 1, although as willbe appreciated by those in the art, the system may not utilize a captureprobe at all (i.e. n is zero), although this is generally not preferred.The upper limit of n will depend on the length of the target sequenceand the required sensitivity. FIG. 3B depicts the use of a singlecapture extender probe 110 with a first portion 111 that will hybridizeto a first portion of the target sequence 120 and a second portion thatwill hybridize to the capture probe 100. FIG. 3C depicts the use of twocapture extender probes 110 and 130. The first capture extender probe110 has a first portion 111 that will hybridize to a first portion ofthe target sequence 120 and a second portion 112 that will hybridize toa first portion 102 of the capture probe 100. The second captureextender probe 130 has a first portion 132 that will hybridize to asecond portion of the target sequence 120 and a second portion 131 thatwill hybridize to a second portion 101 of the capture probe 100. As willbe appreciated by those in the art, any of these attachmentconfigurations may be used with the embodiments of FIGS. 4, 5 and 6.

FIGS. 4A, 4B, 4C and 4D depict several possible mechanism-1 systems.FIGS. 4A, 4B and 4C depict several possible nucleic acid systems. InFIG. 4A, a detection probe 140 (which also serves as a capture probe) isattached to electrode 105 via a conductive oligomer 108. The electrode105 further comprises a monolayer of passivation agents 107. The targetsequence 120 hybridizes to the detection probe 140, and an ETM 135 isattached (either covalently to one or other of the target sequence orthe detection probe or noncovalently, i.e. as a hybridizationindicator). In FIG. 4B, a FIG. 3A attachment to the electrode is used,with a capture probe 100 attached to the electrode 105 using anattachment linker 106, which can be either an insulator or a conductiveoligomer. The target sequence 120 is hybridized to the capture probe,and a label probe 145, comprising a first portion 141 that hybridizes toa portion of the target sequence 105 and a recruitment linker 142 thathybridizes to a detection probe 140 which is attached to the electrodevia a conductive oligomer 108. FIG. 4C is similar, except a firstcapture extender probe 110 is shown, and an amplifier probe 150,comprising a first portion 152 that will hybridize to a second portionof the target sequence 120 and a second portion (amplification sequence)152 that will hybridize to a first portion 141 of the label probe 145. Asecond portion 142 of the label probe 145 hybridizes to the detectionprobe 140, with at least one ETM 135 present. FIG. 3C utilizes a FIG. 3Battachment to the electrode. FIG. 4D depicts a non-nucleic targetanalyte 165 bound to a capture binding ligand 160, attached to theelectrode 105 via an attachment linker 106. A solution binding ligand170 also binds to the target analyte and comprises a recruitment linker171 comprising nucleic acid that will hybridize to the detection probe140 with at least one ETM 135 present.

FIGS. 5A, 5B, 5C, 5D and 5E depict some of the nucleic acid mechanism-2embodiments of the invention. All of the monolayers depicted herein showthe presence of both conductive oligomers 108 and insulators 107 inroughly a 1:1 ratio, although as discussed herein, a variety ofdifferent ratios may be used, or the insulator may be completely absent.In addition, as will be appreciated by those in the art, any one ofthese structures may be repeated for a particular target sequence; thatis, for long target sequences, there may be multiple assay complexesformed. Additionally, any of the electrode-attachment embodiments ofFIG. 3 may be used in any of these systems.

FIGS. 5A, 5B and 5D have the target sequence 120 containing the TMS 135;as discussed herein, these may be added enzymatically, for exampleduring a PCR reaction using nucleotides modified with ETMs, resulting inessentially random incorporation throughout the target sequence, oradded to the terminus of the target sequence. FIG. 5C depicts the use oftwo different capture probes 100 and 100′, that hybridize to differentportions of the target sequence 120. As will be appreciated by those inthe art, the 5′-3′ orientation of the two capture probes in thisembodiment is different.

FIG. 5C depicts the use of label probes 145 that hybridize directly tothe target sequence 120. FIG. 5C shows the use of a label probe 145,comprising a first potion 141 that hybridizes to a portion of the targetsequence 120, a second portion 142 comprising ETMs 135.

FIGS. 5E, 5F and 5G depict systems utilizing label probes 145 that donot hybridize directly to the target, but rather to amplifier probesthat are directly (FIG. 5E) or indirectly (FIGS. 5F and 5G) hybridizedto the target sequence. FIG. 5E utilizes an amplifier probe 150 has afirst portion 151 that hybridizes to the target sequence 120 and atleast one second portion 152, i.e. the amplifier sequence, thathybridizes to the first portion 141 of the label probe. FIG. 5F issimilar, except that a first label extender probe 160 is used,comprising a first portion 161 that hybridizes to the target sequence120 and a second portion 162 that hybridizes to a first portion 151 ofamplifier probe 150. A second portion 152 of the amplifier probe 150hybridizes to a first portion 141 of the label probe 140, which alsocomprises a recruitment linker 142 comprising ETMs 135. FIG. 5G adds asecond label extender probe 170, with a first portion 171 thathybridizes to a portion of the target sequence 120 and a second portionthat hybridizes to a portion of the amplifier probe.

FIG. 5H depicts a system that utilizes multiple label probes. The firstportion 141 of the label probe 140 can hybridize to all or part of therecruitment linker 142.

FIGS. 6A-6H depict some of the possible non-nucleic acid mechanism-2embodiments. FIG. 6A utilizes a capture binding ligand 200 linked to theelectrode 105 by an attachment linker 106. Target analyte 205 binds tothe capture binding ligand 200 and to a solution binding ligand 210 witha recruitment linker 220 comprising ETMs 135. FIG. 6B depicts a similarcase, except that the capture binding ligand 200 is attached to thesurface using a second binding partner interaction, for example anucleic acid; a portion 201 of the capture binding ligand will bind orhybridize to a capture probe 100 on the surface. FIG. 6C also utilizes asecond binding interaction, for example a nucleic acid interaction, toamplify the signal. In this case, the solution binding ligand 210comprises a first portion 220 that will bind or hybridize to a firstporition 231 of a label probe 230. The label probe 230 also comprises asecond portion 232 that is a recruitment linker. FIG. 6D depicts anembodiment similar to 6A, with the use of a second solution bindingligand 240. FIG. 6E depicts the case where more than one capture bindingligand (200 and 200′) is used. FIG. 6F shows a conformation wherein theaddition of target alters the conformation of the binding ligands,causing the recruitment linker 220 to be placed near the monolayersurface. FIG. 6G shows the use of the present invention in candidatebioactive agent screening, wherein the addition of a drug candidate totarget causes the solution binding ligand to dissociate, causing a lossof signal. In addition, the solution binding ligand may be added toanother surface and be bound, as is generally depicted in FIG. 6H forenzymes. FIG. 6H depicts the use of an enzyme to cleave a substratecomprising a recruitment linker, causing a loss of signal. The cleavedpiece may also be added to an additional electrode, causing an increasein signal, using either a mechanism-i or a mechanism-2 system.

FIGS. 7A, 7B, 7C, 7D and 7E depict different possible configurations oflabel probes and attachments of ETMs. In FIGS. 7A-C, the recruitmentlinker is nucleic acid; in FIGS. 7D and E, it is not. A=nucleosidereplacement; B=attachment to a base; C=attachment to a ribose;D=attachment to a phosphate; E=metallocene polymer (although asdescribed herein, this can be a polymer of other ETMs as well), attachedto a base, ribose or phosphate (or other backbone analogs); F=dendrimerstructure, attached via a base, ribose or phosphate (or other backboneanalogs); G=attachment via a “branching” structure, through base, riboseor phosphate (or other backbone analogs); H=attachment of metallocene(or other ETM) polymers; I=attachment via a dendrimer structure;J=attachment using standard linkers.

FIGS. 8A and 8B depicts a schematic of an alternate method of addinglarge numbers of ETMs simultaneously to a nucleic acid using a “branch”point phosphoramidite, as is known in the art. As will be appreciated bythose in the art, each end point can contain any number of ETMs.

FIGS. 9A-C depicts a schematic of the synthesis of simultaneousincorporation of multiple ETMs into a nucleic acid, using a “branch”point nucleoside.

FIG. 10 depicts the synthesis of a “branch” point (in this case anadenosine), to allow the addition of ETM polymers.

FIG. 11 depicts the use of an activated carboxylate for the addition ofa nucleic acid functionalized with a primary amine to a pre-formed SAM.

FIG. 12 depicts a representative hairpin structure. 500 is a targetbinding sequence, 510 is a loop sequence, 520 is a self-complementaryregion, 530 is substantially complementary to a detection probe, and 530is the “sticky end”, that is, a portion that does not hybridize to anyother portion of the probe, that contains the ETMs.

FIGS. 13A, 13B, 13C and 13D depict some embodiments of the invention.

FIG. 14 depicts the results of the experimental example.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to methods and compositions useful inthe detection of biological target analyte species such as nucleic acidsand proteins based on electrochemical detection on an electrode. As isknown in the art, one of the significant hurdles of biosensors,particularly biosensors for the detection of nucleic acids, is the rateof binding (i.e. hybridization in the case of nucleic acids) of thesolution-based target to the surface-bound capture ligand. See forexample Gingeras et al., Nucl. Acid Res. 13:5373 (1987), herebyincorporated by reference in its entirety. This can be affected in anumber of ways, including (1) concentrating the target analyte near thesurface, effectively resulting in a larger amount of the target analytebinding to the capture ligand; (2) configuring the system to allow forgood flow or “mixing”, again allowing a larger amount of the targetanalyte to bind to the capture ligand; or, (3) in the case of nucleicacids, using hybridization accelerators, that actually increase the rateof hybridization in assay complexes comprising the target sequence andthe capture probes. All three of these are sometimes referred to hereinas binding or hybridization acceleration, with the understanding thatsome of these techniques don't actually increase the rate constant ofbinding, they increase the amount of target analyte bound per unit timeby increasing the concentration or by improving mass transport. Thus,the present invention is directed to the use of compositions and methodsto increase the number of target molecules bound to the surface within agiven unit of time. While a number of the techniques outlined herein aregenerally exemplified by nucleic acids, one of skill in the art willrecognize the applicability of all the techniques to other targetanalytes including proteins.

Thus, the present invention describes a number of techniques that can beused to accelerate the rate of assay complex formation or increase thenumber of assay complexes in a given period of time, wherein the targetanalyte becomes associated with a capture ligand on the electrodesurface. These techniques include, but are not limited to,electrophoretic transport; the use of volume exclusion agents; the useof nucleic acid binding proteins (in the case of nucleic acid targetanalytes); the use of polyvalent ions; precipitation agents;partitioning; adjusting the phase compatability; structuring flowparameters; the use of microparticles (including both magnetic andnon-magnetic particles) as either “shuttles” or “mixers”; the use oftemperature gradients; the use of filters; and combinations thereof.

Accordingly, the present invention provides methods of detecting atarget analyte in sample solutions. As will be appreciated by those inthe art, the sample solution may comprise any number of things,including, but not limited to, bodily fluids (including, but not limitedto, blood, urine, serum, lymph, saliva, anal and vaginal secretions,perspiration and semen, of virtually any organism, with mammaliansamples being preferred and human samples being particularly preferred);environmental samples (including, but not limited to, air, agricultural,water and soil samples); biological warfare agent samples; researchsamples (i.e. in the case of nucleic acids, the sample may be theproducts of an amplification reaction, including both target and signalamplification as is generally described in PCT/US99/01705, such as PCRamplification reaction); purified samples, such as purified genomic DNA,RNA, proteins, etc.; raw samples (bacteria, virus, genomic DNA, etc.; Aswill be appreciated by those in the art, virtually any experimentalmanipulation may have been done on the sample.

The methods are directed to the detection of target analytes. By “targetanalytes” or grammatical equivalents herein is meant any molecule orcompound to be detected. As outlined below, target analytes preferablybind to binding ligands, as is more fully described below. As will beappreciated by those in the art, a large number of analytes may bedetected using the present methods; basically, any target analyte forwhich a binding ligand, described below, may be made may be detectedusing the methods of the invention.

When electrophoresis is used, as is more fully outlined below, thetarget analyte is preferably charged, i.e. it carries a net charge underthe experimental conditions, such that it is able to be transportedelectrophoretically in an electric field. However, non-charged targetanalytes may be utilized if a charged binding partner or binding ligandis associated with the target analyte. For example, as more fullydescribed below, in the case of some target analytes, for exampleproteins, that carry little or no net charge, soluble binding ligandscan be used to bind to the target analytes that additionally containETMs and/or charged species; in some embodiments, the ETM may becharged, and thus facilitate both electrophoresis and detection.

Suitable analytes include organic and inorganic molecules, includingbiomolecules. In a preferred embodiment, the analyte may be anenvironmental pollutant (including pesticides, insecticides, toxins,etc.); a chemical (including solvents, organic materials, etc.);therapeutic molecules (including therapeutic and abused drugs,antibiotics, etc.); biomolecules (including hormones, cytokines,proteins, lipids, carbohydrates, cellular membrane antigens andreceptors (neural, hormonal, nutrient, and cell surface receptors) ortheir ligands, etc); whole cells (including procaryotic (such aspathogenic bacteria) and eucaryotic cells, including mammalian tumorcells); viruses (including retroviruses, herpesviruses, adenoviruses,lentiviruses, etc.); and spores; etc. Particularly preferred analytesare environmental pollutants; nucleic acids; proteins (includingenzymes, antibodies, antigens, growth factors, cytokines, etc);therapeutic and abused drugs; cells; and viruses.

Particularly preferred target analytes include proteins and nucleicacids. “Protein” as used herein includes proteins, polypeptides, andpeptides. The protein may be made up of naturally occurring amino acidsand peptide bonds, or synthetic peptidomimetic structures. The sidechains may be in either the (R) or the (S) configuration. In thepreferred embodiment, the amino acids are in the (S) or L-configuration.If non-naturally occurring side chains are used, non-amino acidsubstituents may be used, for example to prevent or retard in vivodegradations.

By “nucleic acid” or “oligonucleotide” or grammatical equivalents hereinmeans at least two nucleotides covalently linked together. A nucleicacid of the present invention will generally contain phosphodiesterbonds, although in some cases, as outlined below, nucleic acid analogsare included that may have alternate backbones, comprising, for example,phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) andreferences therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl etal., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res.14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al.,J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,J. Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamidite linkages (seeEckstein, Oligonucleotides and Analogues: A Practical Approach, OxfordUniversity Press), and peptide nucleic acid backbones and linkages (seeEgholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed.Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al.,Nature 380:207 (1996), all of which are incorporated by reference).Other analog nucleic acids include those with positive backbones (Denpcyet al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones(U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423(1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsingeret al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASCSymposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al.,Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J.Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins etal., Chem. Soc. Rev. (1995) pp169-176). Several nucleic acid analogs aredescribed in Rawls, C & E News Jun. 2, 1997 page 35. All of thesereferences are hereby expressly incorporated by reference. Thesemodifications of the ribose-phosphate backbone may be done to facilitatethe addition of ETMs, or to increase the stability and half-life of suchmolecules in physiological environments.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention. In addition, mixtures ofnaturally occurring nucleic acids and analogs can be made; for example,at the site of conductive oligomer or ETM attachment, an analogstructure may be used. Alternatively, mixtures of different nucleic acidanalogs, and mixtures of naturally occuring nucleic acids and analogsmay be made.

Particularly preferred are peptide nucleic acids (PNA) which includespeptide nucleic acid analogs. These backbones are substantiallynon-ionic under neutral conditions, in contrast to the highly chargedphosphodiester backbone of naturally occurring nucleic acids. Thisresults in two advantages. First, the PNA backbone exhibits improvedhybridization kinetics. PNAs have larger changes in the meltingtemperature (Tm) for mismatched versus perfectly matched basepairs. DNAand RNA typically exhibit a 24° C. drop in Tm for an internal mismatch.With the non-ionic PNA backbone, the drop is closer to 7-9° C. Thisallows for better detection of mismatches. Similarly, due to theirnon-ionic nature, hybridization of the bases attached to these backbonesis relatively insensitive to salt concentration.

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xanthinehypoxanthine, isocytosine, isoguanine, etc. A preferred embodimentutilizes isocytosine and isoguanine in nucleic acids designed to becomplementary to other probes, rather than target sequences, as thisreduces non-specific hybridization, as is generally described in U.S.Pat. No. 5,681,702. As used herein, the term “nucleoside” includesnucleotides as well as nucleoside and nucleotide analogs, and modifiednucleosides such as amino modified nucleosides. In addition,“nucleoside” includes non-naturally occuring analog structures. Thus forexample the individual units of a peptide nucleic acid, each containinga base, are referred to herein as a nucleoside.

In a preferred embodiment, the methods include concentrating the targetanalyte in the vicinity of a detection electrode. The description of thedetection electrode compositions is described below. As will beappreciated by those in the art, the starting concentration of thetarget analyte in the sample can vary widely, depending on the type ofsample used. In general, the starting concentration of the targetanalyte in the sample is relatively low, and preferred techniquesutilize methods that allow the concentration of the target analyte inthe vicinity of the detection electrode.

In general, “concentration” means that the effective diffusion distancea target analyte must travel to bind to the surface is reduced. In apreferred embodiment, the concentration at or near the detectionelectrode is higher than the concentration in the starting sample. Thismay be measured in a variety of ways, including directly, or indirectlyas a function of binding acceleration. That is, in a preferredembodiment, concentration increases of at least two fold are preferred,with at least 5 fold being particularly preferred, and at least 10 foldincreases being especially preferred. As will be appreciated by those inthe art, the increase in concentration will depend on the startingsample size as well, and thus very large increases in concentration,e.g. 100-, 1000- and 10,000- (or higher) fold increases may bedesirable. When the rate of hybridization is used as an indication ofconcentration, increases of at least two fold more target analytebinding to the detection electrode per unit time is preferred, with atleast 5 fold being particularly preferred, and at least 10 foldincreases being especially preferred; again, higher increases may bepreferable in some embodiments.

As outlined herein, there are a variety of suitable concentrationmethods. In a preferred embodiment, the concentrating is done usingelectrophoresis. In general, the system is described as follows. A firstelectrode and a second electrode are used to generate an electric fieldto effect transport, generally electrophoretic transport, of the targetanalyte species increases its concentration at a detection electrode,which has a covalently attached capture binding ligand that will bind(either directly or indirectly ) the target analyte. In this way, thekinetics of target analyte binding to its capture ligand aresignificantly increased, by both increasing the concentration of thetarget analyte in the medium surrounding the capture ligand and reducingthe distance a given target analyte molecule must difuse to find abinding ligand.

The detection electrode may or may not be the same as the firstelectrode. That is, in one embodiment, the electrodes used to generatethe electric fields that result in transport of the analytes to thesurface are different from the electrodes used for detection; i.e. thereare two sets of electrodes, although as will be appreciated by those inthe art, the two sets may share electrodes, for example the counterelectrode. In an additional embodiment, the electrodes used forelectrophoretic transport and for detection are the same; i.e. there isonly one set of electrodes. In some embodiments, the electrophoreticelectrode which attracts the target analyte comprises a permeation layerthat serves to limit access of the target analytes to the electrodesurface and thus protects the analytes from electrochemical degradation.

This electrophoretic transport to the vicinity of the detection probeallows the concentration of the target analyte at or near the detectionprobe surface, which contains capture binding ligands that will bind thetarget analytes to form assay complexes. In some embodiments, thesequential or simultaneous use of a plurality of electrophoresiselectrodes allows multidimensional electrophoresis, i.e. the solutionmay be targeted, “mixed” or “stirred” in the vicinity of the detectionelectrode, to further increase the kinetics of binding. As describedbelow, the assay complex comprises an ETM, which is then detected usingthe detection electrode.

It should also be noted that a number of electrophoretic steps may beused; for example, the components of the system may be addedsequentially, with an electrophoresis step after each addition totransport the reagents down to the detection electrode. Similarly,electrophoresis may be used to effect “washing” steps, wherein excessreagents (non-bound target molecules or non-bound extra binding ligandcomponents, etc.) Or other components of the sample (e.g.noncomplimentary nucleic acids) are driven away from the detectionelectrode. Thus any combination of electrophoresis steps may be used. Inaddition, the time of the electrophoretic steps may be altered.

The methods and compositions of the invention can rely on either twosets of electrodes, wherein one set is used for electrophoresis and thesecond set is used for detection, or one set of electrodes thatfunctions to effect both electrophoresis and detection, as is generallydescribed below.

Samples containing target analytes are placed in an electric fieldbetween at least a first and at least a second electrophoresiselectrode. By “electrode” herein is meant a composition, which, whenconnected to an electronic device, is able to sense a current or apotential and convert it to a signal. Alternatively an electrode can bedefined as a composition which can apply a potential to and/or passelectrons to or from species in the solution. Thus, an electrode is anETM as described below. Preferred electodes are known in the art andinclude, but are not limited to, certain metals and their oxides,including gold; platinum; palladium; silicon; aluminum; metal oxideelectrodes including platinum oxide, titanium oxide, tin oxide, indiumtin oxide, palladium oxide, silicon oxide, aluminum oxide, molybdenumoxide (Mo₂O₆), tungsten oxide (WO₃) and ruthenium oxides; and carbon(including glassy carbon electrodes, graphite and carbon paste).Preferred electrodes include gold, silicon, platinum, carbon and metaloxide electrodes, with gold being particularly preferred.

The electrodes described herein are depicted as a flat surface, which isonly one of the possible conformations of the electrode and is forschematic purposes only. The conformation of the electrode will varywith the detection method used. For example, flat planar electrodes maybe preferred for optical detection methods, or when arrays of nucleicacids are made, thus requiring addressable locations for both synthesisand detection. Alternatively, for single probe analysis, the electrodemay be in the form of a tube, with the SAMs comprising conductiveoligomers and nucleic acids bound to the inner surface. Electrode coilsor mesh may be preferred in some embodiments as well. This allows amaximum of surface area containing the nucleic acids to be exposed to asmall volume of sample.

In addition, as is more fully outlined below, the detection electrodemay be configured to maximize the contact the entire sample has with theelectrode, or to allow mixing, etc.

In a preferred embodiment, one (or both) of the electrophoreticelectrodes comprises a permeation layer, as is generally described inU.S. Pat. Nos. 5,632,957 and 5,605,662, both of which are expresslyincorporated by reference in their entirety. This is particularly usefulwhen the system is run at high voltages, i.e. where water hydrolysisoccurs. The permeation layer serves as an intermediate diffusion layer,and generally has a pore limit property which inhibits or impedes thetarget analytes, reactants, etc. from physical contact with theelectrode surface and thus protects against adverse electrochemicaleffects. The permeation layer may be formed of a variety of materials,including, but not limited to, carbon chain polymers, carbon-siliconchain polymers, carbon-phosphorus chain polymers, carbon-nitrogen chainpolymers, silicon chain polymers, polymer alloys, layered polymercomposites, interpenetrating polymer materials, ceramics, controlledporosity glasss, materials formed as sol-gels, materials formed asaero-gels, agarose, acrylamides, materials formed as hydro-gels, porousgraphite, clays or zeolites. Particularly preferred are mesh-typepolymers formed of acrylamide and cross-linkers, including, but notlimited to, triethylene glycol diacrylate, tetraethylene glycoldiacrylate and N,N′-methylene-bisacrylamide.

The electric field is generated between the first and secondelectrophoresis electrodes. The terms “first” and “second” areessentially interchangeable and not meant to confer any spatial orconformational distinctions, although in general, as used herein, thefirst electrode is generally the electrode spatially closest to thedetection electrode (when two sets of electrodes are used), oralternatively, the first electrode is generally depicted as thedetection electrode (when only one set of electrodes is used). As willbe appreciated by those in the art, any number of possibleelectrophoresis electrode configurations can be used, as is generallydepicted in FIGS. 1 and 2. In general, there are two types ofconfigurations: bulk electrophoresis and targeted electrophoresis.

In a preferred embodiment, a bulk electrophoresis configuration is used.That is, one set of electrophoresis electrodes are used, as is generallyshown in FIGS. 1A, 1B and 1C. The first electrophoresis electrode (10 inFIG. 1) is generally larger than the detection electrodes and isarranged spatially such that the detection probes are within theelectric field generated by the electrophoresis electrodes. Upon theapplication of a DC voltage between the electrodes, an electric field isgenerated such that electrophoretic transport of the charged targetanalytes to the vicinity of the detection probes is effected. While thisdoes not necessarily directly place the target analytes on the detectionprobes, the decrease in effective diffusion distance and increase in theeffective concentration at the detection surface significantly increasesthe kinetics of target analyte binding to the capture binding ligand onthe surface of the detection probe, as diffusion needs to take place inessentially two dimensions, rather than three.

In a preferred embodiment, a targeted electrophoresis configuration isused, as is generally depicted in FIGS. 1D, 1E and 1F. In thisembodiment, there are a plurality of electrophoresis electrodes that areused to specifically target the analyte to a specific detectionelectrode, most generally, but not always, in sets. This may be done inone of two basic ways. In a preferred embodiment, as generally depictedin FIG. 1F and components of which are described in U.S. Pat. No.5,605,662, hereby expressly incorporated by reference, each detectionelectrode has an associated electrophoresis electrode. Thus, by eithersequentially or simultaneously applying a voltage between sets ofelectrophoresis electrodes, target analytes may be moved from onedetection electrode to another. Assuming for the moment a negativelycharged target analyte such as nucleic acid, the system may be run asfollows, using the FIG. 1F system. In one embodiment, an electric fieldis generated between anode 50 and electrophoresis electrode 10, which isacting as the cathode, to bring the anionic target analyte mixture downto both electophoresis electrode 10 and thus detection electrode 20,wherein binding of a species of the target analyte mixture can occur.The electric field is then shut off, and a new electric field is appliedbetween electrophoresis electrode 10, now acting as an anode, andelectrophoresis electrode 11, acting as a cathode. This drives thenon-bound anionic species from 10 to 11, wherein binding of a secondspecies of target analyte can bind. The electric field is turned off,and a new field as between 11 (acting as the anode) and 12 (acting asthe cathode) is generated, to move non-bound target analytes to a newdetection electrode, etc. The advantage of this type of approach is thatessentially the entire target analyte population is transported to eachcapture ligand, thus maximizing the number of target analytes that havean opportunity to bind to their capture ligands.

Alternatively, the electrophoresis at each pad can be runsimultaneously, with an electric field generated between (assuming anegatively charged target analyte population) anode 50 and cathodes 10,11, 12 and 13. This is faster, but results in (assuming four pads) onlyone quarter of the target analyte being “presented” to each detectionelectrode. This may or may not be desirable in different embodiments;for example, when speed rather than sensitivity is important.

In a preferred embodiment, a related but different type of targetedelectrophoresis is done. In this embodiment, a plurality of sets ofelectrophoresis electrodes are positioned in a three dimensional way, toallow movement of the target analytes to different locations. Forexample, as shown in FIG. 2, the use of a three-dimensional array ofelectrophoresis electrodes allows localization of the sample solution toparticular locations, i.e. individual detection electrodes or sets ofdetection electrodes. Thus, for example, with reference to FIG. 2,electrophoretic voltage applied between electrophoretic electrodes 10and 15 and, at the same time, electrophoretic electrodes 12 and 17 candrive the target analyte to detector electrode 20.

Alternatively, in a preferred embodiment, a plurality of electrophoresiselectrodes are used, not for specific targeting to a particularlocation, but rather to increase binding kinetics through “mixing” or“stirring” of the sample in the vicinity of the detection electrode withits associated capture ligand. For example, as shown in FIG. 2, aninitial electrophoretic step may be done between electrophoreticelectrode 18 and a non-depicted second electrophoretic electrode, todrive the target analytes to the detection probe surface, i.e. “bulkelectrophoresis” as defined above. Then, voltages, either DC or ACvoltages, including pulses of each, can be applied between theadditional sets of electrophoresis electrodes, to transport the targetanalyte to increase both availability and binding kinetics of the targetanalytes to the capture binding ligands immobilized on the detectionelectrodes.

The strength of the applied electric field is determined by a number offactors, including, but not limited to, the desired time ofelectrophoresis, the size of the sample (i.e. the distance the targetanalyte must travel), the composition of the solution (i.e. the presenceor absence of electroactive charge carriers and their redox potentials),the composition of the components (i.e. the stability of certaincomponents of the invention to electrochemical potential), the presenceor absence of electroactive charge carriers in solution, the size of thechamber, the charge of the target analyte, the size and location of theelectrodes, the electrode material, etc.

In general, DC voltages are applied for the initial electrophoresis,with DC or AC pulses or fields applied for mixing, if applicable.

The strength of the applied field will depend in part on the othercomponents of the system. For example, when only one set of electrodesis used and thiol linkages are used to attach the components of thesystem to the detection electrodes (i.e. the attachment of passivationagents and conductive oligomers to the detection electrode, as is morefully outlined below), the applied electrophoretic voltage is below theoxidation potential of the thiol compounds, i.e. generally less than 1V. Alternatively, higher voltages can be used when thiol linkages arenot used. Similarly, thiol linkages are acceptable at higher fieldstrengths when two sets of electrodes are used, i.e. the detectionelectrodes containing sensitive chemistry are not exposed to highvoltages.

Thus, in general, electrophoretic voltages range from 1 mV to about 2 V,although as will be appreciated by those in the art, the requiredvoltage will depend on the desired time of running, the net charge onthe analytes, the presence or absence of buffers, the position of theelectrodes, etc. As is known in the art, the electrophoretic velocity isμ(dφ/dx), wherein μ is the ionic mobility. For one set of electrodeembodiments, the electrophoretic voltages range from about 50 mV toabout 900 mV, with from about 100 mV to about 800 mV being preferred,and from about 250 mV to about 700 mV being especially preferred. Fortwo set embodiments, the electrophoretic voltages range from about 100mV to about 2V or higher, with from about 500 mV to about 1.5 V beingpreferred, and from about 1V to about 1.5 V being especially preferred.Of course, as will be appreciated by those in the art, these voltagescan be positive or negative, depending on the charge of the analyte.

When low voltages are used, i.e. voltages less than 1.23 V (relative toa normal hydrogen electrode), the voltage at which water hydrolysisoccurs, it is necessary to include a electroactive species in thesolution, such that current can be transported from the electrode to thesolution and an electric field can be generated throughout the solution.The type and concentration of the electroactive species will vary withthe voltage, the length of required time for electrophoresis (whichrelates to the required distance, i.e. sample volume), etc. In apreferred embodiment, the redox potential of the electroactive speciesis higher than that of the ETM used for detection. For example, if aelectroactive charge carrier with a redox potential of 300 mV is usedand the ETM has a redox potential of 100 mV, electrophoresis can be doneat 300 mV with subsequent detection being done at 100 mV, without a needto remove the electroactive species.

As will be appreciated by those in the art, a wide variety of suitableelectroactive charge carriers can be used, including, but not limitedto, compounds of iron, including aqueous FeCl, Fe(CN)₆ ^(4-/3-), andferrocene and its derivatives; complexes of ruthenium, includingRu(NH₃)₅pyr and Ru(NH₃)₅H₂O; complexes of cobalt including Co(NH₃)₆ ³⁺,Co(bpy)₃ ³⁺ and Co(tris)bpy³⁺); complexes of osmium including Os(bpy)₃²⁺ and Os(tris)bpy and derivatives; complexes of rhenium, includingRh(NH₃)₄Cl₂; and iodine I³⁻. As is known in the art, someoxidation-reduction reactions produce solids (i.e. there are not areversible couple). Generally, these species are not preferred, althoughin some instances they may be used when the detection electrode is thesoluble reaction. Thus for example, a Ag/AgCl reaction may be used withnucleic acids, since the AgCl reaction occurs at the anode. Theconcentration of the redox molecule will vary, as will be appreciated bythose in the art, with concentrations at or below saturation beinguseful. It should also be noted that in this embodiment, when anelectroactive charge carrier is used, it may be necessary to mix or stirthe system during electrophoresis.

It should be noted that electroactive charge carriers may be used in anysystem, particularly array-based systems, that utilize electrophoresis.For example, electroactive charge carriers may be used inelectrophoretic array systems such as described in U.S. Pat. Nos.5,532,129, 5,605,662, 5,565,322 and 5,632,957 and related applications,all of which are incorporated by reference. The sample is placed in theelectric field to effect electrophoretic transport to one or moredetection electrodes. The composition of the detection electrode is asdescribed above for the electrophoresis electrodes, with gold, silicon,carbon, platinum and metal oxide electrodes being particularlypreferred.

In a preferred embodiment, the detection electrodes are formed on asubstrate. In addition, the discussion herein is generally directed tothe formation of gold electrodes, but as will be appreciated by those inthe art, other electrodes can be used as well. The substrate cancomprise a wide variety of materials, as will be appreciated by those inthe art, with printed circuit board (PCB) materials being particularlypreferred. Thus, in general, the suitable substrates include, but arenot limited to, fiberglass, teflon, ceramics, glass, silicon, mica,plastic (including acrylics, polystyrene and copolymers of styrene andother materials, polypropylene, polyethylene, polybutylene,polycarbonate, polyurethanes, Teflon™, and derivatives thereof, etc.),GETEK (a blend of polypropylene oxide and fiberglass), etc.

In general, preferred materials include printed circuit board materials.Circuit board materials are those that comprise an insulating substratethat is coated with a conducting layer and processed using lithographytechniques, particularly photolithography techniques, to form thepatterns of electrodes and interconnects (sometimes referred to in theart as interconnections or leads). The insulating substrate isgenerally, but not always, a polymer. As is known in the art, one or aplurality of layers may be used, to make either “two dimensional” (e.g.all electrodes and interconnections in a plane) or “three dimensional”(wherein the electrodes are on one surface and the interconnects may gothrough the board to the other side) boards. Three dimensional systemsfrequently rely on the use of drilling or etching, followed byelectroplating with a metal such as copper, such that the “throughboard” interconnections are made. Circuit board materials are oftenprovided with a foil already attached to the substrate, such as a copperfoil, with additional copper added as needed (for example forinterconnections), for example by electroplating. The copper surface maythen need to be roughened, for example through etching, to allowattachment of the adhesion layer.

In some embodiments, glass may not be preferred as a substrate.

Accordingly, in a preferred embodiment, the present invention providesbiochips (sometimes referred to herein “chips”) that comprise substratescomprising a plurality of electrodes, preferably gold electrodes. Thenumber of electrodes is as outlined for arrays. Each electrodepreferably comprises a self-assembled monolayer as outlined herein. In apreferred embodiment, one of the monolayer-forming species comprises acapture ligand as outlined herein. In addition, each electrode has aninterconnection, that is attached to the electrode at one end and isultimately attached to a device that can control the electrode. That is,each electrode is independently addressable.

The substrates can be part of a larger device comprising a detectionchamber that exposes a given volume of sample to the detectionelectrode. Generally, the detection chamber ranges from about 1 nL to 1ml, with about 10 μL to 500 μL being preferred. As will be appreciatedby those in the art, depending on the experimental conditions and assay,smaller or larger volumes may be used.

In some embodiments, the detection chamber and electrode are part of acartridge that can be placed into a device comprising electroniccomponents (an AC/DC voltage source, an ammeter, a processor, a read-outdisplay, temperature controller, light source, etc.). In thisembodiment, the interconnections from each electrode are positioned suchthat upon insertion of the cartridge into the device, connectionsbetween the electrodes and the electronic components are established.

Detection electrodes on circuit board material (or other substrates) aregenerally prepared in a wide variety of ways. In general, high puritygold is used, and it may be deposited on a surface via vacuum depositionprocesses (sputtering and evaporation) or solution deposition(electroplating or electroless processes). When electroplating is done,the substrate must initially comprise a conductive material; fiberglasscircuit boards are frequently provided with copper foil. Frequently,depending on the substrate, an adhesion layer between the substrate andthe gold in order to insure good mechanical stability is used. Thus,preferred embodiments utilize a deposition layer of an adhesion metalsuch as chromium, titanium, titanium/tungsten, tantalum, nickel orpalladium, which can be deposited as above for the gold. Whenelectroplated metal (either the adhesion metal or the electrode metal)is used, grain refining additives, frequently referred to in the tradeas brighteners, can optionally be added to alter surface depositionproperties. Preferred brighteners are mixtures of organic and inorganicspecies, with cobalt and nickel being preferred.

In general, the adhesion layer is from about 100 Å thick to about 25microns (1000 microinches). The If the adhesion metal iselectrochemically active, the electrode metal must be coated at athickness that prevents “bleed-through”; if the adhesion metal is notelectrochemically active, the electrode metal may be thinner. Generally,the electrode metal (preferably gold) is deposited at thicknessesranging from about 500 Å to about 5 microns (200 microinches), with fromabout 30 microinches to about 50 microinches being preferred. Ingeneral, the gold is deposited to make electrodes ranging in size fromabout 5 microns to about 5 mm in diameter, with about 100 to 250 micronsbeing preferred. The detection electrodes thus formed are thenpreferably cleaned and SAMs added, as is discussed below.

Thus, the present invention provides methods of making a substratecomprising a plurality of gold electrodes. The methods first comprisecoating an adhesion metal, such as nickel or palladium (optionally withbrightener), onto the substrate. Electroplating is preferred. Theelectrode metal, preferably gold, is then coated (again, withelectroplating preferred) onto the adhesion metal. Then the patterns ofthe device, comprising the electrodes and their associatedinterconnections are made using lithographic techniques, particularlyphotolithographic techniques as are known in the art, and wet chemicaletching. Frequently, a non-conductive chemically resistive insulatingmaterial such as solder mask or plastic is laid down using thesephotolithographic techniques, leaving only the electrodes and aconnection point to the leads exposed; the leads themselves aregenerally coated.

The methods continue with the addition of SAMs. In a preferredembodiment, drop deposition techniques are used to add the requiredchemistry, i.e. the monolayer forming species, one of which ispreferably a capture ligand comprising species. Drop depositiontechniques are well known for making “spot” arrays. This is done to adda different composition to each electrode, i.e. to make an arraycomprising different capture ligands. Alternatively, the SAM species maybe identical for each electrode, and this may be accomplished using adrop deposition technique or the immersion of the entire substrate or asurface of the substrate into the solution.

When a two electrode system is used, a preferred embodiment utilizes aporous detection electrode positioned between the first and the secondelectrophoresis electrodes, such that the target must “go through” thedetection electrode, and thus maximizes the contact of the targetanalyte and the detection electrode. For example, polycarbonatemicroporous membranes are gold sputter coated for electron microscopeanalysis. Alternatively, gold coated polyaniline can be used. Thus, goldelectrodes with very uniform pore sizes, ranging from 0.01 to 20 um canbe made. Similarly, silicon wafers with 10 um pores have been developedfor use as a genosensor that enhance capture of the target sequence by700 fold. By adjusting the flow rate, pad area, pore diameter, depth,and electrophoretic parameters, virtually 100 percent of the targetanalyte in the sample may be bound to the detection electrode.

It should also be noted that a number of electrophoretic steps may beused; for example, the components of the system may be addedsequentially, with an electrophoresis step after each addition totransport the reagents down to the detection electrode. Similarly,electrophoresis may be used to effect “washing” steps, wherein excessreagents (non-bound target molecules or non-bound extra binding ligandcomponents, etc.) are driven away from the detection electrode. Thus anycombination of electrophoresis steps may be used. In addition, the timeof the electrophoretic steps may be altered.

In addition, as is outlined herein, electrophoresis steps can becombined with other techniques to concentrate analytes at the detectionelectrode surface. For example, as is shown in FIG. 13, the system canbe configured to allow flow of the sample past the detection electrodein one direction, coupled with electrophoretic flow in the oppositedirection, thus effectively concentrating the target analyte whileallowing other sample components (particularly uncharged components orcomponents with a charge opposite to the analyte) to be “washed” away.In this embodiment, the strength of the electrophoretic field isadjusted based on the size and charge of the target, such that thetarget remains relatively immobilized at the detection electrode.

In a preferred embodiment, concentration of the target analyte isaccomplished using at least one volume exclusion agent in the assayreagent mix. In this embodiment, the inclusion of a volume exclusionagent, which can absorb solvent and small molecules such as ions, butexcludes larger molecules such as target analytes, concentrates thetarget analyte to smaller apparent volumes, and thus decreasing theeffective diffusional volume that the target analyte experiences, thusincreasing the likelihood of the target finding a capture ligand. Aswill be appreciated by those in the art, the volume exclusion agents maynot necessarily concentrate the sample close to the detection electrode;rather, they decrease the effective diffusional volume that the targetanalyte experiences or resides within.

Thus, the methods of the invention include adding at least one volumeexclusion agent to the array mixture. As will be appreciated by those inthe art, this can be done at virtually any step of the assay, includingpremixture of the exclusion agent with the sample, prior to the additionof additional reagents (such as label probes, etc.), the addition of theexclusion agent with one or more other assay reagents, or after theaddition of the assay reagents. Alternatively, the detection chamber maybe precoated with a volume exclusion agent (for example agarose,sephadex, sepharose, polyacrylamide, etc.) that will swell in thepresence of the sample. In some embodiments, a membrane impermeable tothe target analyte may be used to separate the volume exclusion agentfrom the detection electrode can be used. Similarly, other components ofthe system may be coated with swellable volume exclusion agents, such asthe magnetic particles described herein. In general, adding the agentwith the other assay reagents is preferable. Once added, there isgenerally an incubation step as will be appreciated by those in the art.

Suitable volume exclusion agents are known in the art, and include, butare not limited to, dextran, dextran sulfate, chonchritin sulfate,polyethylene glycol, polysulfonate, heparin sulfate, hespan, highmolecular weight nucleic acid, etc. See for example Amasino, Anal. Chem.152:304 (1986); Wetmur, Biopolymers 14:2517 (1975); Renz et al., Nucl.Acid Res. 12:3435 (1984); Wahl et al., PNAS USA 75:3683 (1979); andGingeras et al., Nucl. Acid Res. 15:5373 (1987), all of which areexpressly incorporated by reference. In addition, as will be appreciatedby those in the art, mixtures of these agents may also be done. Itshould be noted that while volume exclusion is a concentration step, butalso that the exclusion agent may be considered a hybridizationaccelerator, as outlined below.

In a preferred embodiment, concentration of the target analyte is doneby precipitating the target analyte. This is particularly effective fornucleic acids. As has been shown previously, precipitation of nucleicacids can increase the rate of hybridization by 50 to 100 fold; see EP 0229 442 A1, hereby expressly incorporated by reference. As above,precipitation is a concentration step, but the precipitating agent maybe considered a hybridization accelerator, as outlined below. In apreferred embodiment, conditions are selected that precipitate doublestranded nucleic acid but not single stranded nucleic acid, as thisprovides a strong driving force and cuts down on non-specific losses.

Suitable nucleic acid precipitating agents include, but are not limitedto, salts which contain at least one of the stronger salting out cationor anion groups (including the alkali metal salts and ammonium salts ofSO₄, PO₄, Li and COOH); organic compounds that are miscible with thereaction solution and which have precipitating or salting outproperties, including but not limited to, detergent (see Pontius et al.,PNAS USA 88:8237 (1991), hereby incorporated by reference),dihydroxybenezene, Sarkosyl (N-laurosarconsine sodium salt), sodiumdodecyl sulfate, sodium diisobutyl sulfosuccinate and sodium tetradecylsulfate. Suitable concentrations of each agent are described in theincorporated references. It should be noted that in some applications asoutlined below, detergents need not actually precipitate the nucleicacid but rather are added as hybridization accelerators.

In addition, as described in EP 0 229 442 A1, additional reagents may beadded, including, but not limited to, EGTA, EDTA, SDS, SK, PK, EtOH,urea, guanidine HCl, glycogen and dilute amphyl. Furthermore, knownconcentrations of at least one nucleic acid denaturing agents such asalcohol may be added.

As above, the addition of the nucleic acid precipitating agent can bedone at virtually any step of the assay, including premixture of theagent with the sample, prior to the addition of additional reagents(such as label probes, etc.), the addition of the agent with one or moreother assay reagents, or after the addition of the assay reagents. Ingeneral, adding the agent with the other assay reagents is preferable.Again, once added, there is generally an incubation step as will beappreciated by those in the art.

In a preferred embodiment, the concentrating is done by including atleast two reagents that form two separable solution phases, such thatthe target analyte concentrates in one of the phases or at theinterface. As is known in the art, if a sample is subjected to twoseparable solution phases an analyte may be driven from one phase toanother and therefore become concentrated in one phase, or, in somecircumstances, concentration can occur at the interface between the twophases. See for example Albertsson et al., Biochimica et Biophysica Acta103:1-12 (1965), Kohne et al., Biochem. 16(24):5329 (1977), Müller,Partitioning of Nucleic Acids, Ch. 7 in Partitioning in AqueousTwo-Phase Systems, Academic Press, 1985), and Müller et al., Anal.Biochem. 118:269 (1981), all of which are expressly incorporated byreference. Thus, by configuring the sample volume, the volume of eachphase, the detection electrode chamber and the position of the detectionelectrode, good concentration at the electrode may be achieved. As shownin Müller, Partitioning of Nucleic Acids, Ch. 7 in Partitioning inAqueous Two-Phase Systems, Academic Press, (1985) and Albertsson, supra,partitioning is effected by electrolyte composition, including both theionic strength and the kinds of ions, polymer concentration, the size ofthe nucleic acids, the structure and/or complexity of the nucleic acids,and the presence or absence of certain ligands.

In a preferred embodiment, ligands can be included in the partitioningmixtures to effect partitioning. As shown in both Müller et al.references, the inclusion of ligands that bind to nucleic acids caneffect partitioning. Thus for example the use of nucleic acid bindingdyes covalently bound to heteroalkyl chains such as PEG can stronglyraise the partition coefficients. See Müller et al., Anal. Biochem.118:269 (1981), Müller et al., Anal. Biochem. 118:267 (1981); and Mülleret al., Eur. J. Biochem. 128:231 (1982), all of which are expresslyincorporated by reference.

In a preferred embodiment, the phenol emulsion reassociation technique(PERT) is done, as described in Kohne et al., supra. In this embodiment,phenol and water (or other aqueous solutions) are added in the rightproportions and shaken or mixed, an emulsion forms. When the shakingstops, the emulsion breaks and two phases form. The addition of singlestranded nucleic acid and salt to the aqueous phase results in theextremely fast formation of hybrids. As outlined in Kohne, supra, therate of nucleic acid hybridization depends on (a) the presence of theemulsion; (b) the type and concentration of ions; (c) an appropriatetemperature of incubation; (d) the proper pH; (e) the rate and manner ofagitating the emulsion; (f) the amount of phenol present; (g) thefragment size of the nucleic acid; (h) the complexity of the nucleicacid; and (I) the concentration of the nucleic acid.

In addition, partitioning is also known for proteins; see Gineitis etal., Anal. Biochem. 139:400 (1984), hereby expressly incorporated byreference.

In a preferred embodiment, both volume exclusion and partitioning may bedone simultaneously. For example, dextran (3-8%) and PEG (3.5 to 6%) canbe mixed and form separate phases. Shifts in the ratios of cations oranions can transfer high molecular weight nucleic acids from one phaseto another and induce duplex formation.

In a preferred embodiment, the concentration step is done using shuttleparticles. In general, this technique may be described as follows.Shuttle particles that will either settle by gravity onto the detectionelectrode (for example when the detection electrode is at the “bottom”of the chamber), float (for example when the detection electrode is atthe “top” of the chamber) or can be induced to associate with thedetection electrode (for example through the use of magnetic particles)are used. These shuttle particles comprise binding ligands that willassociate with the target analyte(s) in the assay solution, generallybut not always non-specifically, and then shuttle the target analytes tothe detection electrode, where they can be released to bind to thecapture binding ligand (either directly or indirectly, as outlinedbelow). As will be appreciated by those in the art, the shuttle bindingligands preferably interact less strongly with the target analytes thanthe components of the assay complex, i.e. the capture binding ligand.That is, the interaction with the shuttle particle must be weak enoughto allow release of the target analytes for binding to the detectionelectrode.

In a preferred embodiment, the target analyte is a nucleic acid and theshuttle particles may be configured in a number of ways. In one system,the shuttle particles comprise generally short (i.e. 4 to 10, althoughdepending on the temperature used, they may be longer) nucleic acidprobes that can bind the target analytes. These may be either specific,i.e. contain short sequences specific to the target analyte(s) ofinterest, or non-specific, i.e. random probes that will shuttle all thenucleic acid in the sample to the surface. The attachment of nucleicacids to particles is known; see for example U.S. Ser. No. 60/105,875.Similarly, for non-nucleic acid targets, ligands with varying bindingaffinities can be used; for example, a weakly binding antibody to aprotein target may be attached to the bead, and a stronger affinityantibody may serve as the capture ligand on the surface. Alternatively,solution changes may be used to drive the transfer from the bead to thesurface.

Alternatively, for both nucleic acids and other types of analytesincluding proteins, the particles may be modified (for example byderivativization with amine moieties (such as lysine moieties) orcarboxy groups) to contain a charge for the electrostatic interaction ofthe target and the particle.

In a preferred embodiment, again when the target analytes are nucleicacids, the shuttle particles may contain nucleic acid bindingcomponents, that bind to either single stranded or double strandednucleic acids. For example, particles comprising intercalators areknown; see U.S. Pat. No. 5,582,984, hereby incorporated by reference inits entirety. Similarly, particles comprising single-stranded ordouble-stranded binding proteins can be made. Once at the detectionsurface, the target analytes may be released using known techniques,including heat, pH changes, salt changes, etc. It should be noted thatthese particles may have use in sample preparation, as the particles canbind up all the target analyte, allowing the remaining sample to bewashed away or removed, or the particles comprising the target analyteto be removed from the sample.

In addition, as is more fully described below, particles may also beused as “mixing particles”, that serve to stir the solution near thedetection electrode and thus increase hybridization.

In a preferred embodiment, the binding acceleration is done byconfiguring the system to maximize the amount of target analyte that canbind to the detection electrode in a given time period. This may be donefor example by flowing or exposing a large volume of sample containingthe target analyte past the detection electrode such that the targetanalytes have a high probability of associating with the detectionelectrode.

Accordingly, in a preferred embodiment, the methods include flowing thesample containing the target analyte(s) past a detection electrode toform assay complexes. In this embodiment, the concentration of thetarget analyte occurs as a result of a large volume of sample beingcontacted with the detection electrode per unit time, and also decreasesthe binding times as compared to a stagnant sample. Thus, in a preferredembodiment, as outlined above for electrophoresis, the device comprisingthe detection electrode can be configured to have the sample flow pastor through the detection electrode. Thus, a preferred embodimentutilizes a porous electrode such as a gold electrode, as outlined above,positioned in a sample flow channel. See for example WO95/11755,incorporated by reference. The sample may additionally be recirculatedas necessary. Rotating disc electrodes are also preferred.

Thus, in a preferred embodiment, the detection electrode and surroundingarea is configured to result in mixing of the sample, which can serve todisturb this diffusion layer and allow greater access to the surface.For example, in one embodiment, the detection electrode is placed in anarrow sample channel. Thus, essentially, the detection electrode is aband or zone around the perimeter of the channel. Again, as outlinedabove, recirculation can also occur.

In an alternative embodiment, the detection electrode is configured withrespect to the chamber such that the flow of the sample past theelectrode causes mixing or sample turbulence. For example, in oneembodiment the detection electrode is “sunken” or “recessed” withrespect to the chamber, such that the flow of the sample past theelectrode causes mixing; see FIG. 13. This effect may be enhanced byincluding raised surfaces, sometimes referred to herein as “weirs”, onthe edges of the electrode (including sunken electrodes) that causemixing.

In addition to or instead of any of the methods disclosed herein, apreferred embodiment utilizes particles as “mixing balls”. By “particle”or “microparticle” or “nanoparticle” or “bead” or “microsphere” hereinis meant microparticulate matter. As will be appreciated by those in theart, the particles can comprise a wide variety of materials depending ontheir use, including, but not limited to, cross-linked starch, dextrans,cellulose, proteins, organic polymers including styrene polymersincluding polystyrene and methylstyrene as well as other styreneco-polymers, plastics, glass, ceramics, acrylic polymers, magneticallyresponsive materials, colloids, thoria sol, carbon graphite, titaniumdioxide, nylon, latex, and teflon may all be used. “MicrosphereDetection Guide” from Bangs Laboratories, Fishers Ind. is a helpfulguide. Preferred embodiments utilize magnetic particles as outlinedbelow. In addition, in some instances, the mixing particle need notcomprise microparticulate matter; for example, for gravity mixing (i.e.for mixing based on agitation of the device), any component with adensity different from the sample can be used; air bubbles can be usedfor example as mixing particles.

In other embodiments, the mixing particles may be chosen to have a largedielectric constant such that the particles can be moved by theapplication of an electromagnetic field gradient as could be producedusing focused light or a diverging radio frequency (rf) field. Theparticles could also, in some instances, comprising diamagneticmaterials, Such particles would not be affected substantially by theapplication of a linear magnetic field but could be moved by theapplication of a non-linear magnetic field as might be applied using anon-linear magnet or using a large linear magnet combined with smallferro-magnetic or paramagnetic inclusions in the chamber.

The size of the particles will depend on their composition. Theparticles need not be spherical; irregular particles may be used. Inaddition, the particles may be porous, thus increasing the surface areaof the particle available for attachment of moieties. In general, thesize of the particles will vary with their composition; for example,magnetic particles are generally bigger than colloid particles. Thus,the particles have diameters ranging from 1-5 nm (colloids) to 200 μm(magnetic particles).

As will be appreciated by those in the art, the particles can be addedat any point during the assay, including before, during or after theaddition of the sample.

The particles help stir the sample to effect more target binding. Thismay be accomplished in a number of ways. For example, particles can beadded to the detection chamber and the entire chamber or deviceagitated. In a preferred embodiment, magnetic microparticles such as areknown in the art may be used. In a preferred embodiment, the firstparticle is a magnetic particle or a particle that can be induced todisplay magnetic properties. By “magnetic” herein is meant that theparticle is attracted in a magnetic field, including ferromagnetic,paramagnetic, and diamagnetic. In this embodiment, the particles arepreferably from about 0.001 to about 200 μm in diameter, with from about0.05 to about 200 μm preferred, from about 0.1 to about 100 μm beingparticularly preferred, and from about 0.5 to about 10 μm beingespecially preferred.

In this embodiment, it may be preferred to vary the direction and/orstrength of the magnetic field, for example using electromagnetspositioned around the detection chamber to move the beads in a varietyof directions. Thus, for example, the use of magnetic shuttle particlesas both shuttle and mixing particles can be accomplished by multiplemagnetic fields; one that brings the particles down to the detectionelectrode, and one that agitates the beads on the surface of thedetection electrode. Alternatively, non-magnetic particles may be addedto augment the flow-type mixing outlined above.

The size of the microparticles will vary as outlined herein.Microparticles of 4.5 um have been observed to rest in solution on asolid support, relatively unaffected by diffusion, where as in the samesample 1.0 um particles remain suspended away from the solid surface andappear to follow the constraints of diffusion. Thus, the largerparticles may move freely within the diffusion layer when combined withflow, to convert laminar flow to turbulent flow.

In addition, the particles may be chemically altered, for example withvolume exclusion agents or hybridization accelerators as outlined hereinto combine the acceleration effects.

As will be appreciated by those in the art, the shuttle particlesoutlined above may also serve a dual function as mixing particles.

Thus, the present invention provides compositions comprising detectionelectrodes and mixing particles, and methods of detecting targetanalytes using the compositions.

In addition, as is known in the art, one of the rate-limiting steps fortarget capture on a surface is believed to be the diffusion of moleculesacross the boundary layer near the solid phase. This boundary layer doesnot appear to mix well even during flow of the sample. This boundarylayer and its statistical depth is a function of the properties of thesolvent, the solid and the solute. Thus, altering these parameters mayserve to “shrink” the boundary layer the target analyte must pass toreach the surface. For example, adjusting the organic content of thesolute may make the analyte more accessable to the surface. Otherparameters that can effect this are viscosity, surface charge, targetsecondary and tertiary structure, and temperature.

In a preferred embodiment, when the target analyte is a nucleic acid,binding acceleration is done by using a hybridization accelerator. Inthis embodiment, the binding of the target analyte to the detectionelectrode is done in the presence of a hybridization accelerator. Asoutlined herein, there are a variety of hybridization accelerators thatactually increase the rate of nucleic acid hybridization, including, butnot limited to, nucleic acid binding proteins, salts, polyvalent ionsand detergents. In a preferred embodiment, the hybridization acceleratoris a nucleic acid binding protein. As has been shown in the art, certainbinding proteins increase the rate of hybridization of single strandednucleic acids; see Pontius et al., PNAS USA 87:8403 (1990) and U.S. Pat.No. 5,015,569, both of which are incorporated by reference. Thus, forexample, hnRNP (A1 hnRNP) and recA are all known to increase theannealling rate of double stranded nucleic acids. Other single strandednucleic acid binding proteins and major and minor groove bindingproteins may also be used. Suitable conditions are known or elucidatedfrom the prior art.

In a preferred embodiment, the hybridization accelerator is a salt. Asis known in the art, the inclusion of high concentrations of salt canincrease the rate of hybridization; see EP 0 229 442 A1, herebyincorporated by reference. Generally, concentrations of salt up toroughly 2 M can increase the rate of hybridization. Suitable saltsinclude, but are not limited to, sodium chloride, cesium chloride,sodium phosphate, sodium perchlorate, litium chloride, potassiumchloride, sodium bromide, sodium sulfate and ammonium chloride.

In a preferred embodiment, the hybridization accelerator is a polyvalention. Ions of higher valence such as Mg++ can improve the affinity ofnucleic acid strands via electrostatic interactions and thus acceleratehybridization. In addition, these polyvalent ions can potentially affectpackaging on the surface; that is, as the density of nucleic acid on thesurface increases, a negative charge accumulates that may inhibitsubsequent binding of more nucleic acid. Thus, the inclusion of apolyvalent ion that can serve as a “salt bridge” may serve to increasehybridization. Mn++ can have a similar effect.

In addition, certain ions such as Mg++ have been shown to improvebinding in RNA by another method. RNA forms loops aroung Mg++ ions andfinds a stable secondary structure coordinating with Mg++. If Mg++ isremoved the RNA changes to another structure which is also stable.However, the transition phase may be a period of enhanced accessibilityfor incoming probes. Thus, adding sequential rounds of Mg++ followed byEDTA or a similar chelator can cycle through this transition phase andenhance binding.

In a preferred embodiment, the hybridization accelerator is a detergent;see Pontius et al., PNAS USA 88:8237 (1991), hereby incorporated byreference. In this case, certain detergents can increase the rate ofhybridization by as much as 104 fold. Suitable detergents include, butare not limited to, cationic detergents including, but not limited to,dodecyltrimethylammonium bromide (DTAB) and cetyltrimethylammoniumbromide (CTAB), and other variants of the quaternary aminetetramethylammonium bromide (TMAB).

All of the above methods are directed to increasing the amount of targetanalyte accessible for binding and detection on the detection electrodewithin a given period of time. The detection systems of the presentinvention are based on the incorporation of an electron transfer moiety(ETM) into an assay complex as the result of target analyte binding.

In general, there are two basic detection mechanisms. In a preferredembodiment, detection of an ETM is based on electron transfer throughthe stacked π-orbitals of double stranded nucleic acid. This basicmechanism is described in U.S. Pat. Nos. 5,591,578, 5,770,369,5,705,348, and PCT US97/20014 and is termed “mechanism-1” herein.Briefly, previous work has shown that electron transfer can proceedrapidly through the stacked π-orbitals of double stranded nucleic acid,and significantly more slowly through single-stranded nucleic acid.Accordingly, this can serve as the basis of an assay. Thus, by addingETMs (either covalently to one of the strands or non-covalently to thehybridization complex through the use of hybridization indicators,described below) to a nucleic acid that is attached to a detectionelectrode via a conductive oligomer, electron transfer between the ETMand the electrode, through the nucleic acid and conductive oligomer, maybe detected. This general idea is depicted in FIG. 3.

This may be done where the target analyte is a nucleic acid;alternatively, a non-nucleic acid target analyte is used, with anoptional capture binding ligand (to attach the target analyte to thedetection electrode) and a soluble binding ligand that carries a nucleicacid “tail”, that can then bind either directly or indirectly to adetection probe on the surface to effect detection. This general idea isdepicted in FIG. 3C.

Alternatively, the ETM can be detected, not necessarily via electrontransfer through nucleic acid, but rather can be directly detected usingconductive oligomers; that is, the electrons from the ETMs need nottravel through the stacked n orbitals in order to generate a signal.Instead, the presence of ETMs on the surface of a SAM, that comprisesconductive oligomers, can be directly detected. This basic idea istermed “mechanism-2” herein. Thus, upon binding of a target analyte, asoluble binding ligand comprising an ETM is brought to the surface, anddetection of the ETM can proceed. The role of the SAM comprising theconductive oligomers is to shield the electrode from solution componentsand reducing the amount of non-specific binding to the electrodes.Viewed differently, the role of the binding ligand is to providespecificity for a recruitment of ETMs to the surface, where they can bedetected using conductive oligomers with electronically exposed termini.This general idea is shown in FIGS. 4, 5 and 6.

Thus, in either embodiment, as is more fully outlined below, an assaycomplex is formed that contains an ETM, which is then detected using thedetection electrode.

The present system finds particular utility in array formats, i.e.wherein there is a matrix of addressable detection electrodes (hereingenerally referred to “pads”, “addresses” or “micro-locations”). By“array” herein is meant a plurality of capture ligands in an arrayformat; the size of the array will depend on the composition and end useof the array. Arrays containing from about 2 different capture ligandsto many thousands can be made. Generally, the array will comprise fromtwo to as many as 100,000 or more, depending on the size of theelectrodes, as well as the end use of the array. Preferred ranges arefrom about 2 to about 10,000, with from about 5 to about 1000 beingpreferred, and from about 10 to about 100 being particularly preferred.In some embodiments, the compositions of the invention may not be inarray format; that is, for some embodiments, compositions comprising asingle capture ligand may be made as well. In addition, in some arrays,multiple substrates may be used, either of different or identicalcompositions. Thus for example, large arrays may comprise a plurality ofsmaller substrates.

The detection electrode comprises a self-assembled monolayer (SAM). By“monolayer” or “self-assembled monolayer” or “SAM” herein is meant arelatively ordered assembly of molecules spontaneously chemisorbed on asurface, in which the molecules have a preferred orientation relative toeach other (e.g. are oriented approximately parallel to each other) anda preferred orientation relative to the surface (e.g. roughlyperpendicular to it). Each of the molecules includes a functional groupthat adheres to the surface, and a portion that interacts withneighboring molecules in the monolayer to form the relatively orderedarray. A “mixed” monolayer comprises a heterogeneous monolayer, that is,where at least two different molecules make up the monolayer. The SAMmay comprise conductive oligomers alone, or a mixture of conductiveoligomers and insulators. As outlined herein, the efficiency of targetanalyte binding (for example, oligonucleotide hybridization) mayincrease when the analyte is at a distance from the electrode.Similarly, non-specific binding of biomolecules, including the targetanalytes, to an electrode is generally reduced when a monolayer ispresent. Thus, a monolayer facilitates the maintenance of the analyteaway from the electrode surface. In addition, a monolayer serves to keepextraneous electroactive species away from the surface of the electrode.Thus, this layer helps to prevent electrical contact between theelectrodes and the ETMs, or between the electrode and extraneouselectroactive species within the solvent. Such contact can result in adirect “short circuit” or an indirect short circuit via charged specieswhich may be present in the sample. Accordingly, in one embodiment, themonolayer is preferably tightly packed in a uniform layer on theelectrode surface, such that a minimum of “holes” exist. In thisembodiment, the monolayer thus serves as a physical barrier to blocksolvent accesibility to the electrode.

In a preferred embodiment, the monolayer comprises conductive oligomers.By “conductive oligomer” herein is meant a substantially conductingoligomer, preferably linear, some embodiments of which are referred toin the literature as “molecular wires”. By “substantially conducting”herein is meant that the oligomer is capable of transfering electrons at100 Hz. Generally, the conductive oligomer has substantially overlappingπ-orbitals, i.e. conjugated π-orbitals, as between the monomeric unitsof the conductive oligomer, although the conductive oligomer may alsocontain one or more sigma (σ) bonds. Additionally, a conductive oligomermay be defined functionally by its ability to inject or receiveelectrons into or from an associated ETM. Furthermore, the conductiveoligomer is more conductive than the insulators as defined herein.Additionally, the conductive oligomers of the invention are to bedistinguished from electroactive polymers, that themselves may donate oraccept electrons.

In a preferred embodiment, the conductive oligomers have a conductivity,S, of from between about 10⁻⁶ to about 10⁴ Ω⁻¹cm⁻¹, with from about 10⁻⁵to about 10³ Ω⁻¹cm⁻¹ being preferred, with these S values beingcalculated for molecules ranging from about 20 Å to about 200 Å. Asdescribed below, insulators have a conductivity S of about 10⁻⁷ Ω⁻¹cm⁻¹or lower, with less than about 10⁻⁸ Ω⁻¹cm⁻¹ being preferred. Seegenerally Gardner et al., Sensors and Actuators A 51 (1995) 57-66,incorporated herein by reference.

Desired characteristics of a conductive oligomer include highconductivity, sufficient solubility in organic solvents and/or water forsynthesis and use of the compositions of the invention, and preferablychemical resistance to reactions that occur i) during binding ligandsynthesis (i.e. nucleic acid synthesis, such that nucleosides containingthe conductive oligomers may be added to a nucleic acid synthesizerduring the synthesis of the compositions of the invention, ii) duringthe attachment of the conductive oligomer to an electrode, or iii)during binding assays. In addition, conductive oligomers that willpromote the formation of self-assembled monolayers are preferred.

The oligomers of the invention comprise at least two monomeric subunits,as described herein. As is described more fully below, oligomers includehomo- and hetero-oligomers, and include polymers.

In a preferred embodiment, the conductive oligomer has the structuredepicted in Structure 1:

As will be understood by those in the art, all of the structuresdepicted herein may have additional atoms or structures; i.e. theconductive oligomer of Structure 1 may be attached to ETMs, such aselectrodes, transition metal complexes, organic ETMs, and metallocenes,and to binding ligands such as nucleic acids, or to several of these.Unless otherwise noted, the conductive oligomers depicted herein will beattached at the left side to an electrode; that is, as depicted inStructure 1, the left “Y” is connected to the electrode as describedherein. If the conductive oligomer is to be attached to a bindingligand, the right “Y”, if present, is attached to the binding ligandsuch as a nucleic acid, either directly or through the use of a linker,as is described herein.

In this embodiment, Y is an aromatic group, n is an integer from 1 to50, g is either 1 or zero, e is an integer from zero to 10, and m iszero or 1. When g is 1, B—D is a bond able to conjugate with neighboringbonds (herein referred to as a “conjugated bond”), preferably selectedfrom acetylene, alkene, substituted alkene, amide, azo, —C═N— (including—N═C—, —CR═N— and —N═CR—), —Si═Si—, and —Si═C— (including —C═Si—,—Si═CR— and —CR═Si—). When g is zero, e is preferably 1, D is preferablycarbonyl, or a heteroatom moiety, wherein the heteroatom is selectedfrom oxygen, sulfur, nitrogen, silicon or phosphorus. Thus, suitableheteroatom moieties include, but are not limited to, —NH and —NR,wherein R is as defined herein; substituted sulfur; sulfonyl (—SO₂—)sulfoxide (—SO—); phosphine oxide (—PO— and —RPO—); and thiophosphine(—PS— and —RPS—). However, when the conductive oligomer is to beattached to a gold electrode, as outlined below, sulfur derivatives arenot preferred. By “aromatic group” or grammatical equivalents herein ismeant an aromatic monocyclic or polycyclic hydrocarbon moiety generallycontaining 5 to 14 carbon atoms (although larger polycyclic ringsstructures may be made) and any carbocylic ketone or thioketonederivative thereof, wherein the carbon atom with the free valence is amember of an aromatic ring. Aromatic groups include arylene groups andaromatic groups with more than two atoms removed. For the purposes ofthis application aromatic includes heterocycle. “Heterocycle” or“heteroaryl” means an aromatic group wherein 1 to 5 of the indicatedcarbon atoms are replaced by a heteroatom chosen from nitrogen, oxygen,sulfur, phosphorus, boron and silicon wherein the atom with the freevalence is a member of an aromatic ring, and any heterocyclic ketone andthioketone derivative thereof. Thus, heterocycle includes thienyl,furyl, pyrrolyl, pyrimidinyl, oxalyl, indolyl, purinyl, quinolyl,isoquinolyl, thiazolyl, imidozyl, etc.

Importantly, the Y aromatic groups of the conductive oligomer may bedifferent, i.e. the conductive oligomer may be a heterooligomer. Thatis, a conductive oligomer may comprise a oligomer of a single type of Ygroups, or of multiple types of Y groups.

The aromatic group may be substituted with a substitution group,generally depicted herein as R. R groups may be added as necessary toaffect the packing of the conductive oligomers, i.e. R groups may beused to alter the association of the oligomers in the monolayer. Rgroups may also be added to 1) alter the solubility of the oligomer orof compositions containing the oligomers; 2) alter the conjugation orelectrochemical potential of the system; and 3) alter the charge orcharacteristics at the surface of the monolayer.

In a preferred embodiment, when the conductive oligomer is greater thanthree subunits, R groups are preferred to increase solubility whensolution synthesis is done. However, the R groups, and their positions,are chosen to minimally effect the packing of the conductive oligomerson a surface, particularly within a monolayer, as described below. Ingeneral, only small R groups are used within the monolayer, with largerR groups generally above the surface of the monolayer. Thus for examplethe attachment of methyl groups to the portion of the conductiveoligomer within the monolayer to increase solubility is preferred, withattachment of longer alkoxy groups, for example, C3 to C10, ispreferably done above the monolayer surface. In general, for the systemsdescribed herein, this generally means that attachment of stericallysignificant R groups is not done on any of the first two or threeoligomer subunits, depending on the average length of the moleculesmaking up the monolayer.

Suitable R groups include, but are not limited to, hydrogen, alkyl,alcohol, aromatic, amino, amido, nitro, ethers, esters, aldehydes,sulfonyl, silicon moieties, halogens, sulfur containing moieties,phosphorus containing moieties, and ethylene glycols. In the structuresdepicted herein, R is hydrogen when the position is unsubstituted. Itshould be noted that some positions may allow two substitution groups, Rand R′, in which case the R and R′ groups may be either the same ordifferent.

By “alkyl group” or grammatical equivalents herein is meant a straightor branched chain alkyl group, with straight chain alkyl groups beingpreferred. If branched, it may be branched at one or more positions, andunless specified, at any position. The alkyl group may range from about1 to about 30 carbon atoms (C1-C30), with a preferred embodimentutilizing from about 1 to about 20 carbon atoms (C1-C20), with about C1through about C12 to about C15 being preferred, and C1 to C5 beingparticularly preferred, although in some embodiments the alkyl group maybe much larger. Also included within the definition of an alkyl groupare cycloalkyl groups such as C5 and C6 rings, and heterocyclic ringswith nitrogen, oxygen, sulfur or phosphorus. Alkyl also includesheteroalkyl, with heteroatoms of sulfur, oxygen, nitrogen, and siliconebeing preferred. Alkyl includes substituted alkyl groups. By“substituted alkyl group” herein is meant an alkyl group furthercomprising one or more substitution moieties “R”, as defined above.

By “amino groups” or grammatical equivalents herein is meant —NH₂, —NHRand —NR₂ groups, with R being as defined herein.

By “nitro group” herein is meant an —NO₂ group.

By “sulfur containing moieties” herein is meant compounds containingsulfur atoms, including but not limited to, thia-, thio- andsulfo-compounds, thiols (—SH and —SR), and sulfides (—RSR—). By“phosphorus containing moieties” herein is meant compounds containingphosphorus, including, but not limited to, phosphines and phosphates. By“silicon containing moieties” herein is meant compounds containingsilicon.

By “ether” herein is meant an —O—R group. Preferred ethers includealkoxy groups, with —O—(CH₂)₂CH₃ and —O—(CH₂)₄CH₃ being preferred.

By “ester” herein is meant a —COOR group.

By “halogen” herein is meant bromine, iodine, chlorine, or fluorine.Preferred substituted alkyls are partially or fully halogenated alkylssuch as CF₃, etc.

By “aldehyde” herein is meant —RCHO groups.

By “alcohol” herein is meant —OH groups, and alkyl alcohols —ROH.

By “amido” herein is meant —RCONH— or RCONR— groups.

By “ethylene glycol” or “(poly)ethylene glycol” herein is meant a—(O—CH₂—CH₂)_(n)— group, although each carbon atom of the ethylene groupmay also be singly or doubly substituted, i.e. —(O—CR₂—CR₂)_(n)—, with Ras described above. Ethylene glycol derivatives with other heteroatomsin place of oxygen (i.e. —(N—CH₂—CH₂),— or —(S—CH₂—CH₂)_(n)—, or withsubstitution groups) are also preferred.

Preferred substitution groups include, but are not limited to, methyl,ethyl, propyl, alkoxy groups such as —O—(CH₂)₂CH₃ and —O—(CH₂)₄CH₃ andethylene glycol and derivatives thereof.

Preferred aromatic groups include, but are not limited to, phenyl,naphthyl, naphthalene, anthracene, phenanthroline, pyrole, pyridine,thiophene, porphyrins, and substituted derivatives of each of these,included fused ring derivatives.

In the conductive oligomers depicted herein, when g is 1, B—D is a bondlinking two atoms or chemical moieties. In a preferred embodiment, B—Dis a conjugated bond, containing overlapping or conjugated π-orbitals.

Preferred B—D bonds are selected from acetylene (—C═C—, also calledalkyne or ethyne), alkene (—CH═CH—, also called ethylene), substitutedalkene (—CR═CR—, —CH═CR— and —CR═CH—), amide (—NH—CO— and —NR—CO— or—CO—NH— and —CO—NR—), azo (—N═N—), esters and thioesters (—CO—O—,—O—CO—, —CS—O— and —O—CS—) and other conjugated bonds such as (—CH═N—,—CR═N—, —N═CH— and —N═CR—), (—SiH═SiH—, —SiR═SiH—, —SiR═SiH—, and—SiR═SiR—), (—SiH═CH—, —SiR═CH—, —SiH═CR—, —SiR═CR—, —CH═SiH—, —CR═SiH—,—CH═SiR—, and —CR═SiR—). Particularly preferred B-D bonds are acetylene,alkene, amide, and substituted derivatives of these three, and azo.Especially preferred B—D bonds are acetylene, alkene and amide. Theoligomer components attached to double bonds may be in the trans or cisconformation, or mixtures. Thus, either B or D may include carbon,nitrogen or silicon. The substitution groups are as defined as above forR.

When g=0 in the Structure 1 conductive oligomer, e is preferably 1 andthe D moiety may be carbonyl or a heteroatom moiety as defined above.

As above for the Y rings, within any single conductive oligomer, the B—Dbonds (or D moieties, when g=0) may be all the same, or at least one maybe different. For example, when m is zero, the terminal B—D bond may bean amide bond, and the rest of the B—D bonds may be acetylene bonds.Generally, when amide bonds are present, as few amide bonds as possibleare preferable, but in some embodiments all the B—D bonds are amidebonds. Thus, as outlined above for the Y rings, one type of B—D bond maybe present in the conductive oligomer within a monolayer as describedbelow, and another type above the monolayer level, for example to givegreater flexibility for nucleic acid hybridization when the nucleic acidis attached via a conductive oligomer.

In the structures depicted herein, n is an integer from 1 to 50,although longer oligomers may also be used (see for example Schumm etal., Angew. Chem. Int. Ed. Engl. 1994 33(13):1360). Without being boundby theory, it appears that for efficient hybridization of nucleic acidson a surface, the hybridization should occur at a distance from thesurface, i.e. the kinetics of hybridization increase as a function ofthe distance from the surface, particularly for long oligonucleotides of200 to 300 basepairs. Accordingly, when a nucleic acid is attached via aconductive oligomer, as is more fully described below, the length of theconductive oligomer is such that the closest nucleotide of the nucleicacid is positioned from about 6 Å to about 100 Å (although distances ofup to 500 Å may be used) from the electrode surface, with from about 15Å to about 60 Å being preferred and from about 25 Å to about 60 Å alsobeing preferred. Accordingly, n will depend on the size of the aromaticgroup, but generally will be from about 1 to about 20, with from about 2to about 15 being preferred and from about 3 to about 10 beingespecially preferred.

In the structures depicted herein, m is either 0 or 1. That is, when mis 0, the conductive oligomer may terminate in the B—D bond or D moiety,i.e. the D atom is attached to the nucleic acid either directly or via alinker. In some embodiments, for example when the conductive oligomer isattached to a phosphate of the ribose-phosphate backbone of a nucleicacid, there may be additional atoms, such as a linker, attached betweenthe conductive oligomer and the nucleic acid. Additionally, as outlinedbelow, the D atom may be the nitrogen atom of the amino-modified ribose.Alternatively, when m is 1, the conductive oligomer may terminate in Y,an aromatic group, i.e. the aromatic group is attached to the nucleicacid or linker.

As will be appreciated by those in the art, a large number of possibleconductive oligomers may be utilized. These include conductive oligomersfalling within the Structure 1 and Structure 8 formulas, as well asother conductive oligomers, as are generally known in the art, includingfor example, compounds comprising fused aromatic rings or Teflon®-likeoligomers, such as —(CF₂)_(n)—, —(CHF)_(n)— and —(CFR)_(n)—. See forexample, Schumm et al., Angew. Chem. Intl. Ed. Engl. 33:1361(1994);Grosshenny et al., Platinum Metals Rev. 40(1):26-35 (1996); Tour,Chem. Rev. 96:537-553 (1996); Hsung et al., Organometallics 14:4808-4815(1995; and references cited therein, all of which are expresslyincorporated by reference.

Particularly preferred conductive oligomers of this embodiment aredepicted below:

Structure 2 is Structure 1 when g is 1. Preferred embodiments ofStructure 2 include: e is zero, Y is pyrole or substituted pyrole; e iszero, Y is thiophene or substituted thiophene; e is zero, Y is furan orsubstituted furan; e is zero, Y is phenyl or substituted phenyl; e iszero, Y is pyridine or substituted pyridine; e is 1, B—D is acetyleneand Y is phenyl or substituted phenyl (see Structure 4 below). Apreferred embodiment of Structure 2 is also when e is one, depicted asStructure 3 below:

Preferred embodiments of Structure 3 are: Y is phenyl or substitutedphenyl and B—D is azo; Y is phenyl or substituted phenyl and B—D isacetylene; Y is phenyl or substituted phenyl and B—D is alkene; Y ispyridine or substituted pyridine and B—D is acetylene; Y is thiophene orsubstituted thiophene and B—D is acetylene; Y is furan or substitutedfuran and B—D is acetylene; Y is thiophene or furan (or substitutedthiophene or furan) and B—D are alternating alkene and acetylene bonds.

Most of the structures depicted herein utilize a Structure 3 conductiveoligomer. However, any Structure 3 oligomers may be substituted with anyof the other structures depicted herein, i.e. Structure 1 or 8 oligomer,or other conducting oligomer, and the use of such Structure 3 depictionis not meant to limit the scope of the invention.

Particularly preferred embodiments of Structure 3 include Structures 4,5, 6 and 7, depicted below:

Particularly preferred embodiments of Structure 4 include: n is two, mis one, and R is hydrogen; n is three, m is zero, and R is hydrogen; andthe use of R groups to increase solubility.

When the B—D bond is an amide bond, as in Structure 5, the conductiveoligomers are pseudopeptide oligomers. Although the amide bond inStructure 5 is depicted with the carbonyl to the left, i.e. —CONH—, thereverse may also be used, i.e. —NHCO—. Particularly preferredembodiments of Structure include: n is two, m is one, and R is hydrogen;n is three, m is zero, and R is hydrogen (in this embodiment, theterminal nitrogen (the D atom) may be the nitrogen of the amino-modifiedribose); and the use of R groups to increase solubility.

Preferred embodiments of Structure 6 include the first n is two, secondn is one, m is zero, and all R groups are hydrogen, or the use of Rgroups to increase solubility.

Preferred embodiments of Structure 7 include: the first n is three, thesecond n is from 1-3, with m being either 0 or 1, and the use of Rgroups to increase solubility.

In a preferred embodiment, the conductive oligomer has the structuredepicted in Structure 8:

In this embodiment, C are carbon atoms, n is an integer from 1 to 50, mis 0 or 1, J is a heteroatom selected from the group consisting ofoxygen, nitrogen, silicon, phosphorus, sulfur, carbonyl or sulfoxide,and G is a bond selected from alkane, alkene or acetylene, such thattogether with the two carbon atoms the C—G—C group is an alkene(—CH═CH—), substituted alkene (—CR═CR—) or mixtures thereof (—CH═CR— or—CR═CH—), acetylene (—C═C—), or alkane (—CR₂—CR₂—, with R being eitherhydrogen or a substitution group as described herein). The G bond ofeach subunit may be the same or different than the G bonds of othersubunits; that is, alternating oligomers of alkene and acetylene bondscould be used, etc. However, when G is an alkane bond, the number ofalkane bonds in the oligomer should be kept to a minimum, with about sixor less sigma bonds per conductive oligomer being preferred. Alkenebonds are preferred, and are generally depicted herein, although alkaneand acetylene bonds may be substituted in any structure or embodimentdescribed herein as will be appreciated by those in the art.

In some embodiments, for example when ETMs are not present, if m=0 thenat least one of the G bonds is not an alkane bond.

In a preferred embodiment, the m of Structure 8 is zero. In aparticularly preferred embodiment, m is zero and G is an alkene bond, asis depicted in Structure 9:

The alkene oligomer of structure 9, and others depicted herein, aregenerally depicted in the preferred trans configuration, althougholigomers of cis or mixtures of trans and cis may also be used. Asabove, R groups may be added to alter the packing of the compositions onan electrode, the hydrophilicity or hydrophobicity of the oligomer, andthe flexibility, i.e. the rotational, torsional or longitudinalflexibility of the oligomer. n is as defined above.

In a preferred embodiment, R is hydrogen, although R may be also alkylgroups and polyethylene glycols or derivatives.

In an alternative embodiment, the conductive oligomer may be a mixtureof different types of oligomers, for example of structures 1 and 8.

The conductive oligomers may or may not have terminal groups. Thus, in apreferred embodiment, there is no additional terminal group, and theconductive oligomer terminates with one of the groups depicted inStructures 1 to 9; for example, a B—D bond such as an acetylene bond.Alternatively, in a preferred embodiment, a terminal group is added,sometimes depicted herein as “Q”. A terminal group may be used forseveral reasons; for example, to contribute to the electronicavailability of the conductive oligomer for detection of ETMs, or toalter the surface of the SAM for other reasons, for example to preventnon-specific binding. For example, when the target analyte is a nucleicacid, there may be negatively charged groups on the terminus to form anegatively charged surface such that when the nucleic acid is DNA or RNAthe nucleic acid is repelled or prevented from lying down on thesurface, to facilitate hybridization. Preferred terminal groups include—NH₂, —OH, —COOH, and alkyl groups such as —CH₃, and (poly)alkyloxidessuch as (poly)ethylene glycol, with —OCH₂CH₂OH, —(OCH₂CH₂O)₂H,—(OCH₂CH₂O)₃H, and —(OCH₂CH₂O)₄H being preferred.

In one embodiment, it is possible to use mixtures of conductiveoligomers with different types of terminal groups. Thus, for example,some of the terminal groups may facilitate detection, and some mayprevent non-specific binding.

It will be appreciated that the monolayer may comprise differentconductive oligomer species, although preferably the different speciesare chosen such that a reasonably uniform SAM can be formed. Thus, forexample, when capture binding ligands such as nucleic acids arecovalently attached to the electrode using conductive oligomers, it ispossible to have one type of conductive oligomer used to attach thenucleic acid, and another type in the SAM. Similarly, it may bedesirable to have mixtures of different lengths of conductive oligomersin the monolayer, to help reduce non-specific signals. Thus, forexample, preferred embodiments utilize conductive oligomers thatterminate below the surface of the rest of the monolayer, i.e. below theinsulator layer, if used, or below some fraction of the other conductiveoligomers. Similarly, the use of different conductive oligomers may bedone to facilitate monolayer formation, or to make monolayers withaltered properties.

In a preferred embodiment, the monolayer may further comprise insulatormoieties. By “insulator” herein is meant a substantially nonconductingoligomer, preferably linear. By “substantially nonconducting” herein ismeant that the insulator will not transfer electrons at 100 Hz. The rateof electron transfer through the insulator is preferrably slower thanthe rate through the conductive oligomers described herein.

In a preferred embodiment, the insulators have a conductivity, S, ofabout 10⁻⁷ Ω⁻¹cm⁻¹ or lower, with less than about 10⁻⁸ Ω⁻¹cm⁻¹ beingpreferred. See generally Gardner et al., supra. Generally, insulatorsare alkyl or heteroalkyl oligomers or moieties with sigma bonds,although any particular insulator molecule may contain aromatic groupsor one or more conjugated bonds. By “heteroalkyl” herein is meant analkyl group that has at least one heteroatom, i.e. nitrogen, oxygen,sulfur, phosphorus, silicon or boron included in the chain.Alternatively, the insulator may be quite similar to a conductiveoligomer with the addition of one or more heteroatoms or bonds thatserve to inhibit or slow, preferably substantially, electron transfer.

Suitable insulators are known in the art, and include, but are notlimited to, —(CH₂)_(n)—, —(CRH)_(n)—, and —(CR₂)_(n)—, ethylene glycolor derivatives using other heteroatoms in place of oxygen, i.e. nitrogenor sulfur (sulfur derivatives are not preferred when the electrode isgold).

As for the conductive oligomers, the insulators may be substituted withR groups as defined herein to alter the packing of the moieties orconductive oligomers on an electrode, the hydrophilicity orhydrophobicity of the insulator, and the flexibility, i.e. therotational, torsional or longitudinal flexibility of the insulator. Forexample, branched alkyl groups may be used. Similarly, the insulatorsmay contain terminal groups, as outlined above, particularly toinfluence the surface of the monolayer.

The length of the species making up the monolayer will vary as needed.As outlined above, it appears that binding of target analytes (forexample, hybridization of nucleic acids) is more efficient at a distancefrom the surface. The species to which capture binding ligands areattached (as outlined below, these can be either insulators orconductive oligomers) may be basically the same length as the monolayerforming species or longer than them, resulting in the capture bindingligands being more accessible to the solvent for hybridization. In someembodiments, the conductive oligomers to which the capture bindingligands are attached may be shorter than the monolayer.

As will be appreciated by those in the art, the actual combinations andratios of the different species making up the monolayer can vary widely,and will depend on whether mechanism-1 or -2 is used, and, in the caseof electrophoresis, whether a one electrode system or two electrodesystem is used, as is more fully outlined below. Generally, threecomponent systems are preferred for mechanism-2 systems, with the firstspecies comprising a capture binding ligand containing species (termed acapture probe when the target analyte is a nucleic acid), attached tothe electrode via either an insulator or a conductive oligomer. Thesecond species are conductive oligomers, and the third species areinsulators. In this embodiment, the first species can comprise fromabout 90% to about 1%, with from about 20% to about 40% being preferred.When the target analytes are nucleic acids, from about 30% to about 40%is especially preferred for short oligonucleotide targets and from about10% to about 20% is preferred for longer targets. The second species cancomprise from about 1% to about 90%, with from about 20% to about 90%being preferred, and from about 40% to about 60% being especiallypreferred. The third species can comprise from about 1% to about 90%,with from about 20% to about 40% being preferred, and from about 15% toabout 30% being especially preferred. To achieve these approximateproportions, preferred ratios of first:second:third species in SAMformation solvents are 2:2:1 for short targets, 1:3:1 for longertargets, with total thiol concentration (when used to attach thesespecies, as is more fully outlined below) in the 500 μM to 1 mM range,and 833 μM being preferred.

Alternatively, two component systems can be used. In one embodiment, foruse in either mechanism-1 or mechanism-2 systems, the two components arethe first and second species. In this embodiment, the first species cancomprise from about 1% to about 90%, with from about 1% to about 40%being preferred, and from about 10% to about 40% being especiallypreferred. The second species can comprise from about 1% to about 90%,with from about 10% to about 60% being preferred, and from about 20% toabout 40% being especially preferred. Alternatively, for mechanism-1systems, the two components are the first and the third species. In thisembodiment, the first species can comprise from about 1% to about 90%,with from about 1% to about 40% being preferred, and from about 10% toabout 40% being especially preferred. The second species can comprisefrom about 1% to about 90%, with from about 10% to about 60% beingpreferred, and from about 20% to about 40% being especially preferred.

In a preferred embodiment, the deposition of the SAM is done usingaqueous solvents. As is generally described in Steel et al., Anal. Chem.70:4670 (1998), Herne et al., J. Am. Chem. Soc. 119:8916 (1997), andFinklea, Electrochemistry of Organized Monolayers of Thiols and RelatedMolecules on Electrodes, from A. J. Bard, Electroanalytical Chemistry: ASeries of Advances, Vol. 20, Dekker N. Y. 1966-, all of which areexpressly incorporated by reference, the deposition of the SAM-formingspecies can be done out of aqueous solutions, frequently comprisingsalt.

In addition, when electrophoresis systems are used, the composition andintegrity of the monolayer may depend on whether a one electrode or twoelectrode system is used. Thus, for example, if a one electrode systemis used for both electrophoresis and detection, the configuration of thesystem will allow the electroactive charge carriers, if used, access tothe electrode. As will be appreciated by those in the art, if thechemistry of attachment of the conductive oligomer is stable at the highvoltages used to hydrolyze water, no electroactive charge carriers needbe used. This may be done in one of several ways. In a preferredembodiment, the monolayer comprises a significant component ofelectronically exposed conductive oligomers; a monolayer such as thiseffective raises the surface of the electrode, allowing theelectroactive charge carriers indirect access to the electrode.Alternatively, a poor monolayer may be used, i.e. a monolayer thatcontains “pinholes” or “imperfections”, such that there is directsolvent access to the electrode. Alternatively, the configuration of theelectrode may be such that less than the entire surface of the electrodeis covered by a SAM, to allow direct access to the electrode, butminimizing the surface for non-specific binding.

The covalent attachment of the conductive oligomers and insulators tothe electrode may be accomplished in a variety of ways, depending on theelectrode and the composition of the insulators and conductive oligomersused. In a preferred embodiment, the attachment linkers with covalentlyattached nucleosides or nucleic acids as depicted herein are covalentlyattached to an electrode. Thus, one end or terminus of the attachmentlinker is attached to the nucleoside or nucleic acid, and the other isattached to an electrode. In some embodiments it may be desirable tohave the attachment linker attached at a position other than a terminus,or even to have a branched attachment linker that is attached to anelectrode at one terminus and to two or more nucleosides at othertermini, although this is not preferred. Similarly, the attachmentlinker may be attached at two sites to the electrode, as is generallydepicted in Structures 11-13. Generally, some type of linker is used, asdepicted below as “A” in Structure 10, where “X” is the conductiveoligomer, “I” is an insulator and the hatched surface is the electrode:

In this embodiment, A is a linker or atom. The choice of “A” will dependin part on the characteristics of the electrode. Thus, for example, Amay be a sulfur moiety when a gold electrode is used. Alternatively,when metal oxide electrodes are used, A may be a silicon (silane) moietyattached to the oxygen of the oxide (see for example Chen et al.,Langmuir 10:3332-3337 (1994); Lenhard et al., J. Electroanal. Chem.78:195-201 (1977), both of which are expressly incorporated byreference). When carbon based electrodes are used, A may be an aminomoiety (preferably a primary amine; see for example Deinhammer et al.,Langmuir 10:1306-1313 (1994)). Thus, preferred A moieties include, butare not limited to, silane moieties, sulfur moieties (including alkylsulfur moieties), and amino moieties. In a preferred embodiment, epoxidetype linkages with redox polymers such as are known in the art are notused.

Although depicted herein as a single moiety, the insulators andconductive oligomers may be attached to the electrode with more than one“A” moiety; the “A” moieties may be the same or different. Thus, forexample, when the electrode is a gold electrode, and “A” is a sulfuratom or moiety, multiple sulfur atoms may be used to attach theconductive oligomer to the electrode, such as is generally depictedbelow in Structures 11, 12 and 13. As will be appreciated by those inthe art, other such structures can be made. In Structures 11, 12 and 13,the A moiety is just a sulfur atom, but substituted sulfur moieties mayalso be used.

It should also be noted that similar to Structure 13, it may be possibleto have a a conductive oligomer terminating in a single carbon atom withthree sulfur moities attached to the electrode. Additionally, althoughnot always depicted herein, the conductive oligomers and insulators mayalso comprise a “Q” terminal group.

In a preferred embodiment, the electrode is a gold electrode, andattachment is via a sulfur linkage as is well known in the art, i.e. theA moiety is a sulfur atom or moiety. Although the exact characteristicsof the gold-sulfur attachment are not known, this linkage is consideredcovalent for the purposes of this invention. A representative structureis depicted in Structure 14, using the Structure 3 conductive oligomer,although as for all the structures depicted herein, any of theconductive oligomers, or combinations of conductive oligomers, may beused. Similarly, any of the conductive oligomers or insulators may alsocomprise terminal groups as described herein. Structure 14 depicts the“A” linker as comprising just a sulfur atom, although additional atomsmay be present (i.e. linkers from the sulfur to the conductive oligomeror substitution groups). In addition, Structure 14 shows the sulfur atomattached to the Y aromatic group, but as will be appreciated by those inthe art, it may be attached to the B—D group (i.e. an acetylene) aswell.

In general, thiol linkages are preferred. In systems usingelectrophoresis, thiol linkages are preferred when either two sets ofelectrodes are used (i.e. the detection electrodes comprising the SAMsare not used at high electrophoretic voltages (i.e. greater than 800 or900 mV), that can cause oxidation of the thiol linkage and thus loss ofthe SAM), or, if one set of electrodes is used, lower electrophoreticvoltages are used as is generally described below.

In a preferred embodiment, the electrode is a carbon electrode, i.e. aglassy carbon electrode, and attachment is via a nitrogen of an aminegroup. A representative structure is depicted in Structure 15. Again,additional atoms may be present, i.e. Z type linkers and/or terminalgroups.

In structure 16, the oxygen atom is from the oxide of the metal oxideelectrode. The Si atom may also contain other atoms, i.e. be a siliconmoiety containing substitution groups. Other attachments for SAMs toother electrodes are known in the art; see for example Napier et al.,Langmuir, 1997, for attachment to indium tin oxide electrodes, and alsothe chemisorption of phosphates to an indium tin oxide electrode (talkby H. Holden Thorpe, CHI conference, May 4-5, 1998).

The SAMs of the invention can be made in a variety of ways, includingdeposition out of organic solutions and deposition out of aqueoussolutions. The methods outlined herein use a gold electrode as theexample, although as will be appreciated by those in the art, othermetals and methods may be used as well. In one preferred embodiment,indium-tin-oxide (ITO) is used as the electrode.

In a preferred embodiment, a gold surface is first cleaned. A variety ofcleaning procedures may be employed, including, but not limited to,chemical cleaning or etchants (including Piranha solution (hydrogenperoxide/sulfuric acid) or aqua regia (hydrochloric acid/nitric acid),electrochemical methods, flame treatment, plasma treatment orcombinations thereof.

Following cleaning, the gold substrate is exposed to the SAM species.When the electrode is ITO, the SAM species are phosphonate-containingspecies. This can also be done in a variety of ways, including, but notlimited to, solution deposition, gas phase deposition, microcontactprinting, spray deposition, deposition using neat components, etc. Apreferred embodiment utilizes a deposition solution comprising a mixtureof various SAM species in solution, generally thiol-containing species.Mixed monolayers that contain target analytes, particularly DNA, areusually prepared using a two step procedure. The thiolated DNA isdeposited during the first deposition step (generally in the presence ofat least one other monolayer-forming species) and the mixed monolayerformation is completed during the second step in which a second thiolsolution minus DNA is added. The second step frequently involves mildheating to promote monolayer reorganization.

In a preferred embodiment, the deposition solution is an organicdeposition solution. In this embodiment, a clean gold surface is placedinto a clean vial. A binding ligand deposition solution in organicsolvent is prepared in which the total thiol concentration is betweenmicromolar to saturation; preferred ranges include from about 1 μM to 10mM, with from about 400 uM to about 1.0 mM being especially preferred.In a preferred embodiment, the deposition solution contains thiolmodified DNA (i.e. nucleic acid attached to an attachment linker) andthiol diluent molecules (either conductive oligomers or insulators, withthe latter being preferred). The ratio of DNA to diluent (if present) isusually between 1000:1 to 1:1000, with from about 10:1 to about 1:10being preferred and 1:1 being especially preferred. The preferredsolvents are tetrahydrofuran (THF), acetonitrile, dimethylforamide(DMF), ethanol, or mixtures thereof; generally any solvent of sufficientpolarity to dissolve the capture ligand can be used, as long as thesolvent is devoid of functional groups that will react with the surface.Sufficient DNA deposition solution is added to the vial so as tocompletely cover the electrode surface. The gold substrate is allowed toincubate at ambient temperature or slightly above ambient temperaturefor a period of time ranging from seconds to hours, with 5-30 minutesbeing preferred. After the initial incubation, the deposition solutionis removed and a solution of diluent molecule only (from about 1 μM to10 mM, with from about 100 uM to about 1.0 mM being preferred) inorganic solvent is added. The gold substrate is allowed to incubate atroom temperature or above room temperature for a period of time (secondsto days, with from about 10 minutes to about 24 hours being preferred).The gold sample is removed from the solution, rinsed in clean solventand used.

In a preferred embodiment, an aqueous deposition solution is used. Asabove, a clean gold surface is placed into a clean vial. A DNAdeposition solution in water is prepared in which the total thiolconcentration is between about 1 uM and 10 mM, with from about 1 μM toabout 200 uM being preferred. The aqueous solution frequently has saltpresent (up to saturation, with approximately 1M being preferred),however pure water can be used. The deposition solution contains thiolmodified DNA and often a thiol diluent molecule. The ratio of DNA todiluent is usually between between 1000:1 to 1:1000, with from about10:1 to about 1:10 being preferred and 1:1 being especially preferred.The DNA deposition solution is added to the vial in such a volume so asto completely cover the electrode surface. The gold substrate is allowedto incubate at ambient temperature or slightly above ambient temperaturefor 1-30 minutes with 5 minutes usually being sufficient. After theinitial incubation, the deposition solution is removed and a solution ofdiluent molecule only (10 uM-1.0 mM) in either water or organic solventis added. The gold substrate is allowed to incubate at room temperatureor above room temperature until a complete monolayer is formed (10minutes-24 hours). The gold sample is removed from the solution, rinsedin clean solvent and used.

In a preferred embodiment, as outlined herein, a circuit board is usedas the substrate for the gold electrodes. Formation of the SAMs on thegold surface is generally done by first cleaning the boards, for examplein a 10% sulfuric acid solution for 30 seconds, detergent solutions,aqua regia, plasma, etc., as outlined herein. Following the sulfuricacid treatment, the boards are washed, for example via immersion in twoMilli-Q water baths for 1 minute each. The boards are then dried, forexample under a stream of nitrogen. Spotting of the deposition solutiononto the boards is done using any number of known spotting systems,generally by placing the boards on an X-Y table, preferably in ahumidity chamber. The size of the spotting drop will vary with the sizeof the electrodes on the boards and the equipment used for delivery ofthe solution; for example, for 250 μM size electrodes, a 30 nanoliterdrop is used. The volume should be sufficient to cover the electrodesurface completely. The drop is incubated at room temperature for aperiod of time (sec to overnight, with 5 minutes preferred) and then thedrop is removed by rinsing in a Milli-Q water bath. The boards are thenpreferably treated with a second deposition solution, generallycomprising insulator in organic solvent, preferably acetonitrile, byimmersion in a 45° C. bath. After 30 minutes, the boards are removed andimmersed in an acetonitrile bath for 30 seconds followed by a milli-Qwater bath for 30 seconds. The boards are dried under a stream ofnitrogen.

In a preferred embodiment, the detection electrode further comprises acapture binding ligand, preferably covalently attached. By “bindingligand” or “binding species” herein is meant a compound that is used toprobe for the presence of the target analyte, that will bind to thetarget analyte. In general, for most of the embodiments describedherein, there are at least two binding ligands used per target analytemolecule; a “capture” or “anchor” binding ligand (also referred toherein as a “capture probe”, particularly in reference to a nucleic acidbinding ligand) that is attached to the detection electrode as describedherein, and a soluble binding ligand, that binds independently to thetarget analyte, and either directly or indirectly comprises at least oneETM.

Generally, the capture binding ligand allows the attachment of a targetanalyte to the detection electrode, for the purposes of detection. As ismore fully outlined below, attachment of the target analyte to thecapture binding ligand may be direct (i.e. the target analyte binds tothe capture binding ligand) or indirect (one or more capture extenderligands may be used).

In a preferred embodiment, the binding is specific, and the bindingligand is part of a binding pair. By “specifically bind” herein is meantthat the ligand binds the analyte, with specificity sufficient todifferentiate between the analyte and other components or contaminantsof the test sample. However, as will be appreciated by those in the art,it will be possible to detect analytes using binding that is not highlyspecific; for example, the systems may use different binding ligands,for example an array of different ligands, and detection of anyparticular analyte is via its “signature” of binding to a panel ofbinding ligands, similar to the manner in which “electronic noses” work.The binding should be sufficient to allow the analyte to remain boundunder the conditions of the assay, including wash steps to removenon-specific binding. In some embodiments, for example in the detectionof certain biomolecules, the binding constants of the analyte to thebinding ligand will be at least about 10⁻⁴ to 10⁻⁶ M⁻¹, with at leastabout 10⁻⁵ to 10⁻⁹ being preferred and at least about 10⁻⁷ to 10⁻⁹ M⁻¹being particularly preferred.

As will be appreciated by those in the art, the composition of thebinding ligand will depend on the composition of the target analyte.Binding ligands to a wide variety of analytes are known or can bereadily found using known techniques. For example, when the analyte is asingle-stranded nucleic acid, the binding ligand is generally asubstantially complementary nucleic acid. Alternatively, as is generallydescribed in U.S. Pat. Nos. 5,270,163, 5,475,096, 5,567,588, 5,595,877,5,637,459, 5,683,867, 5,705,337, and related patents, herebyincorporated by reference, nucleic acid “aptomers” can be developed forbinding to virtually any target analyte. Similarly the analyte may be anucleic acid binding protein and the capture binding ligand is either asingle-stranded or double-stranded nucleic acid; alternatively, thebinding ligand may be a nucleic acid binding protein when the analyte isa single or double-stranded nucleic acid. When the analyte is a protein,the binding ligands include proteins (particularly including antibodiesor fragments thereof (FAbs, etc.)), small molecules, or aptamers,described above. Preferred binding ligand proteins include peptides. Forexample, when the analyte is an enzyme, suitable binding ligands includesubstrates, inhibitors, and other proteins that bind the enzyme, i.e.components of a multi-enzyme (or protein) complex. As will beappreciated by those in the art, any two molecules that will associate,preferably specifically, may be used, either as the analyte or thebinding ligand. Suitable analyte/binding ligand pairs include, but arenot limited to, antibodies/antigens, receptors/ligand, proteins/nucleicacids; nucleic acids/nucleic acids, enzymes/substrates and/orinhibitors, carbohydrates (including glycoproteins andglycolipids)/lectins, carbohydrates and other binding partners,proteins/proteins; and protein/small molecules. These may be wild-typeor derivative sequences. In a preferred embodiment, the binding ligandsare portions (particularly the extracellular portions) of cell surfacereceptors that are known to multimerize, such as the growth hormonereceptor, glucose transporters (particularly GLUT4 receptor),transferrin receptor, epidermal growth factor receptor, low densitylipoprotein receptor, high density lipoprotein receptor, leptinreceptor, interleukin receptors including IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-15 and IL-17 receptors,VEGF receptor, PDGF receptor, EPO receptor, TPO receptor, ciliaryneurotrophic factor receptor, prolactin receptor, and T-cell receptors.Similarly, there is a wide body of literature relating to thedevelopment of binding partners based on combinatorial chemistrymethods.

In this embodiment, when the binding ligand is a nucleic acid, preferredcompositions and techniques are outlined in WO 98/20162; PCT/US98/12430;PCT/US98/12082; PCT/US99/1705; PCT/US99/01703; and U.S. Ser. Nos.09/135,183; 60/105,875; and 09/295,691, all of which are herebyexpressly incorporated by reference.

The method of attachment of the capture binding ligands to theattachment linker (either an insulator or conductive oligomer) willgenerally be d one as is known in the art, and will depend on both thecomposition of the attachment linker and the capture binding ligand. Ingeneral, the capture binding ligands are attached to the attachmentlinker through the use of functional groups on each that can then beused for attachment. Preferred functional groups for attachment areamino groups, carboxy groups, oxo groups and thiol groups. Thesefunctional groups can then be attached, either directly or indirectlythrough the use of a linker, sometimes depicted herein as “Z”. Linkersare well known in the art; for example, homo-or hetero-bifunctionallinkers as are well known (see 1994 Pierce Chemical Company catalog,technical section on cross-linkers, pages 155-200, incorporated hereinby reference). Preferred Z linkers include, but are not limited to,alkyl groups (including substituted alkyl groups and alkyl groupscontaining heteroatom moieties), with short alkyl groups, esters, amide,amine, epoxy groups and ethylene glycol and derivatives being preferred, with propyl, acetylene, and C₂ alkene being especiallypreferred. Z may also be a sulfone group, forming sulfonamide linkages.

In this way, capture binding ligands comprising proteins, lectins,nucleic acids, small organic molecules, carbohydrates, etc. can beadded.

A preferred embodiment utilizes proteinaceous capture binding ligands.As is known in the art, any number of techniques may be used to attach aproteinaceous capture binding ligand to an attachment linker. A widevariety of techniques are known to add moieties to proteins.

A preferred embodiment utilizes nucleic acids as the capture bindingligand. While most of the following discussion focuses on nucleic acids,as will be appreciated by those in the art, many of the techniquesoutlined below apply in a similar manner to non-nucleic acid systems aswell.

The capture probe nucleic acid is covalently attached to the electrode,via an “attachment linker”, that can be either a conductive oligomer(required for mechanism-1 systems) or an insulator. By “covalentlyattached” herein is meant that two moieties are attached by at least onebond, including sigma bonds, pi bonds and coordination bonds.

Thus, one end of the attachment linker is attached to a nucleic acid (orother binding ligand), and the other end (although as will beappreciated by those in the art, it need not be the exact terminus foreither) is attached to the electrode. Thus, any of structures depictedherein may further comprise a nucleic acid effectively as a terminalgroup. Thus, the present invention provides compositions comprisingnucleic acids covalently attached to electrodes as is generally depictedbelow in Structure 17:

In Structure 17, the hatched marks on the left represent an electrode. Xis a conductive oligomer and I is an insulator as defined herein. F₁ isa linkage that allows the covalent attachment of the electrode and theconductive oligomer or insulator, including bonds, atoms or linkers suchas is described herein, for example as “A”, defined below. F₂ is alinkage that allows the covalent attachment of the conductive oligomeror insulator to the nucleic acid, and may be a bond, an atom or alinkage as is herein described. F₂ may be part of the conductiveoligomer, part of the insulator, part of the nucleic acid, or exogeneousto both, for example, as defined herein for “Z”.

In a preferred embodiment, the capture probe nucleic acid is covalentlyattached to the electrode via a conductive oligomer. The covalentattachment of the nucleic acid and the conductive oligomer may beaccomplished in several ways. In a preferred embodiment, the attachmentis via attachment to the base of the nucleoside, via attachment to thebackbone of the nucleic acid (either the ribose, the phosphate, or to ananalogous group of a nucleic acid analog backbone), or via a transitionmetal ligand, as described below. The techniques outlined below aregenerally described for naturally occuring nucleic acids, although aswill be appreciated by those in the art, similar techniques may be usedwith nucleic acid analogs, and in some cases with other binding ligands.

In a preferred embodiment, the conductive oligomer is attached to thebase of a nucleoside of the nucleic acid. This may be done in severalways, depending on the oligomer, as is described below. In oneembodiment, the oligomer is attached to a terminal nucleoside, i.e.either the 3′ or 5′ nucleoside of the nucleic acid. Alternatively, theconductive oligomer is attached to an internal nucleoside.

The point of attachment to the base will vary with the base. Generally,attachment at any position is possible. In some embodiments, for examplewhen the probe containing the ETMs may be used for hybridization (i.e.mechanism-1 systems), it is preferred to attach at positions notinvolved in hydrogen bonding to the complementary base. Thus, forexample, generally attachment is to the 5 or 6 position of pyrimidinessuch as uridine, cytosine and thymine. For purines such as adenine andguanine, the linkage is preferably via the 8 position. Attachment tonon-standard bases is preferably done at the comparable positions.

In one embodiment, the attachment is direct; that is, there are nointervening atoms between the conductive oligomer and the base. In thisembodiment, for example, conductive oligomers with terminal acetylenebonds are attached directly to the base. Structure 18 is an example ofthis linkage, using a Structure 3 conductive oligomer and uridine as thebase, although other bases and conductive oligomers can be used as willbe appreciated by those in the art:

It should be noted that the pentose structures depicted herein may havehydrogen, hydroxy, phosphates or other groups such as amino groupsattached. In addition, the pentose and nucleoside structures depictedherein are depicted non-conventionally, as mirror images of the normalrendering. In addition, the pentose and nucleoside structures may alsocontain additional groups, such as protecting groups, at any position,for example as needed during synthesis.

In addition, the base may contain additional modifications as needed,i.e. the carbonyl or amine groups may be altered or protected.

In an alternative embodiment, the attachment is any number of differentZ linkers, including amide and amine linkages, as is generally depictedin Structure 19 using uridine as the base and a Structure 3 oligomer:

In this embodiment, Z is a linker. Preferably, Z is a short linker ofabout 1 to about 10 atoms, with from 1 to 5 atoms being preferred, thatmay or may not contain alkene, alkynyl, amine, amide, azo, imine, etc.,bonds. Linkers are known in the art; for example, homo-orhetero-bifunctional linkers as are well known (see 1994 Pierce ChemicalCompany catalog, technical section on cross-linkers, pages 155-200,incorporated herein by reference). Preferred Z linkers include, but arenot limited to, alkyl groups (including substituted alkyl groups andalkyl groups containing heteroatom moieties), with short alkyl groups,esters, amide, amine, epoxy groups and ethylene glycol and derivativesbeing preferred, with propyl, acetylene, and C₂ alkene being especiallypreferred. Z may also be a sulfone group, forming sulfonamide linkagesas discussed below.

In a preferred embodiment, the attachment of the nucleic acid and theconductive oligomer is done via attachment to the backbone of thenucleic acid. This may be done in a number of ways, including attachmentto a ribose of the ribose-phosphate backbone, or to the phosphate of thebackbone, or other groups of analogous backbones.

As a preliminary matter, it should be understood that the site ofattachment in this embodiment may be to a 3′ or 5′ terminal nucleotide,or to an internal nucleotide, as is more fully described below.

In a preferred embodiment, the conductive oligomer is attached to theribose of the ribose-phosphate backbone. This may be done in severalways. As is known in the art, nucleosides that are modified at eitherthe 2′ or 3′ position of the ribose with amino groups, sulfur groups,silicone groups, phosphorus groups, or oxo groups can be made (Imazawaet al., J. Org. Chem., 44:2039 (1979); Hobbs et al., J. Org. Chem.42(4):714 (1977); Verheyden et al., J. Orrg. Chem. 36(2):250 (1971);McGee et al., J. Org. Chem. 61:781-785 (1996); Mikhailopulo et al.,Liebigs. Ann. Chem. 513-519 (1993); McGee et al., Nucleosides &Nucleotides 14(6):1329 (1995), all of which are incorporated byreference). These modified nucleosides are then used to add theconductive oligomers.

A preferred embodiment utilizes amino-modified nucleosides. Theseamino-modified riboses can then be used to form either amide or aminelinkages to the conductive oligomers. In a preferred embodiment, theamino group is attached directly to the ribose, although as will beappreciated by those in the art, short linkers such as those describedherein for “Z” may be present between the amino group and the ribose.

In a preferred embodiment, an amide linkage is used for attachment tothe ribose. Preferably, if the conductive oligomer of Structures 1-3 isused, m is zero and thus the conductive oligomer terminates in the amidebond. In this embodiment, the nitrogen of the amino group of theamino-modified ribose is the “D” atom of the conductive oligomer. Thus,a preferred attachment of this embodiment is depicted in Structure 20(using the Structure 3 conductive oligomer):

As will be appreciated by those in the art, Structure 20 has theterminal bond fixed as an amide bond.

In a preferred embodiment, a heteroatom linkage is used, i.e. oxo,amine, sulfur, etc. A preferred embodiment utilizes an amine linkage.Again, as outlined above for the amide linkages, for amine linkages, thenitrogen of the amino-modified ribose may be the “D” atom of theconductive oligomer when the Structure 3 conductive oligomer is used.Thus, for example, Structures 21 and 22 depict nucleosides with theStructures 3 and 9 conductive oligomers, respectively, using thenitrogen as the heteroatom, athough other heteroatoms can be used:

In Structure 21, preferably both m and t are not zero. A preferred Zhere is a methylene group, or other aliphatic alkyl linkers. One, two orthree carbons in this position are particularly useful for syntheticreasons.

In Structure 22, Z is as defined above. Suitable linkers includemethylene and ethylene.

In an alternative embodiment, the conductive oligomer is covalentlyattached to the nucleic acid via the phosphate of the ribose-phosphatebackbone (or analog) of a nucleic acid. In this embodiment, theattachment is direct, utilizes a linker or via an amide bond. Structure23 depicts a direct linkage, and Structure 24 depicts linkage via anamide bond (both utilize the Structure 3 conductive oligomer, althoughStructure 8 conductive oligomers are also possible). Structures 23 and24 depict the conductive oligomer in the 3′ position, although the 5′position is also possible. Furthermore, both Structures 23 and 24 depictnaturally occurring phosphodiester bonds, although as those in the artwill appreciate, non-standard analogs of phosphodiester bonds may alsobe used.

In Structure 23, if the terminal Y is present (i.e. m=1), thenpreferably Z is not present (i.e. t=0). If the terminal Y is notpresent, then Z is preferably present.

Structure 24 depicts a preferred embodiment, wherein the terminal B—Dbond is an amide bond, the terminal Y is not present, and Z is a linker,as defined herein.

In a preferred embodiment, the conductive oligomer is covalentlyattached to the nucleic acid via a transition metal ligand. In thisembodiment, the conductive oligomer is covalently attached to a ligandwhich provides one or more of the coordination atoms for a transitionmetal. In one embodiment, the ligand to which the conductive oligomer isattached also has the nucleic acid attached, as is generally depictedbelow in Structure 25. Alternatively, the conductive oligomer isattached to one ligand, and the nucleic acid is attached to anotherligand, as is generally depicted below in Structure 26. Thus, in thepresence of the transition metal, the conductive oligomer is covalentlyattached to the nucleic acid. Both of these structures depict Structure3 conductive oligomers, although other oligomers may be utilized.Structures 25 and 26 depict two representative structures:

In the structures depicted herein, M is a metal atom, with transitionmetals being preferred. Suitable transition metals for use in theinvention include, but are not limited to, cadmium (Cd), copper (Cu),cobalt (Co), palladium (Pd), zinc (Zn), iron (Fe), ruthenium (Ru),rhodium (Rh), osmium (Os), rhenium (Re), platinium (Pt), scandium (Sc),titanium (Ti), Vanadium (V), chromium (Cr), manganese (Mn), nickel (Ni),Molybdenum (Mo), technetium (Tc), tungsten (W), and iridium (Ir). Thatis, the first series of transition metals, the platinum metals (Ru, Rh,Pd, Os, Ir and Pt), along with Fe, Re, W, Mo and Tc, are preferred.Particularly preferred are ruthenium, rhenium, osmium, platinium, cobaltand iron.

L are the co-ligands, that provide the coordination atoms for thebinding of the metal ion. As will be appreciated by those in the art,the number and nature of the co-ligands will depend on the coordinationnumber of the metal ion. Mono-, di- or polydentate co-ligands may beused at any position. Thus, for example, when the metal has acoordination number of six, the L from the terminus of the conductiveoligomer, the L contributed from the nucleic acid, and r, add up to six.Thus, when the metal has a coordination number of six, r may range fromzero (when all coordination atoms are provided by the other two ligands)to four, when all the co-ligands are monodentate. Thus generally, r willbe from 0 to 8, depending on the coordination number of the metal ionand the choice of the other ligands.

In one embodiment, the metal ion has a coordination number of six andboth the ligand attached to the conductive oligomer and the ligandattached to the nucleic acid are at least bidentate; that is, r ispreferably zero, one (i.e. the remaining co-ligand is bidentate) or two(two monodentate co-ligands are used).

As will be appreciated in the art, the co-ligands can be the same ordifferent. Suitable ligands fall into two categories: ligands which usenitrogen, oxygen, sulfur, carbon or phosphorus atoms (depending on themetal ion) as the coordination atoms (generally referred to in theliterature as sigma (a) donors) and organometallic ligands such asmetallocene ligands (generally referred to in the literature as pi (π)donors, and depicted herein as L_(m)). Suitable nitrogen donatingligands are well known in the art and include, but are not limited to,NH₂; NHR; NRR′; pyridine; pyrazine; isonicotinamide; imidazole;bipyridine and substituted derivatives of bipyridine; terpyridine andsubstituted derivatives; phenanthrolines, particularly1,10-phenanthroline (abbreviated phen) and substituted derivatives ofphenanthrolines such as 4,7-dimethylphenanthroline anddipyridol[3,2-a:2′,3′-c]phenazine (abbreviated dppz); dipyridophenazine;1,4,5,8,9,12-hexaazatriphenylene (abbreviated hat);9,10-phenanthrenequinone diimine (abbreviated phi);1,4,5,8-tetraazaphenanthrene (abbreviated tap);1,4,8,11-tetra-azacyclotetradecane (abbreviated cyclam), EDTA, EGTA andisocyanide. Substituted derivatives, including fused derivatives, mayalso be used. In some embodiments, porphyrins and substitutedderivatives of the porphyrin family may be used. See for example,Comprehensive Coordination Chemistry, Ed. Wilkinson et al., PergammonPress, 1987, Chapters 13.2 (pp73-98), 21.1 (pp. 813-898) and 21.3 (pp915-957), all of which are hereby expressly incorporated by reference.

Suitable sigma donating ligands using carbon, oxygen, sulfur andphosphorus are known in the art. For example, suitable sigma carbondonors are found in Cotton and Wilkenson, Advanced Organic Chemistry,5th Edition, John Wiley & Sons, 1988, hereby incorporated by reference;see page 38, for example. Similarly, suitable oxygen ligands includecrown ethers, water and others known in the art. Phosphines andsubstituted phosphines are also suitable; see page 38 of Cotton andWilkenson.

The oxygen, sulfur, phosphorus and nitrogen-donating ligands areattached in such a manner as to allow the heteroatoms to serve ascoordination atoms.

In a preferred embodiment, organometallic ligands are used. In additionto purely organic compounds for use as redox moieties, and varioustransition metal coordination complexes with δ-bonded organic ligandwith donor atoms as heterocyclic or exocyclic substituents, there isavailable a wide variety of transition metal organometallic compoundswith π-bonded organic ligands (see Advanced Inorganic Chemistry, 5thEd., Cotton & Wilkinson, John Wiley & Sons, 1988, chapter 26;Organometallics, A Concise Introduction, Elschenbroich et al., 2nd Ed.,1992, VCH; and Comprehensive Organometallic Chemistry II, A Review ofthe Literature 1982-1994, Abel et al. Ed., Vol. 7, chapters 7, 8, 10 &11, Pergamon Press, hereby expressly incorporated by reference). Suchorganometallic ligands include cyclic aromatic compounds such as thecyclopentadienide ion [C₅H₅(−1)] and various ring substituted and ringfused derivatives, such as the indenylide (−1) ion, that yield a classof bis(cyclopentadieyl) metal compounds, (i.e. the metallocenes); seefor example Robins et al., J. Am. Chem. Soc. 104:1882-1893 (1982); andGassman et al., J. Am. Chem. Soc. 108:4228-4229 (1986), incorporated byreference. Of these, ferrocene [(C₅H₅)₂Fe] and its derivatives areprototypical examples which have been used in a wide variety of chemical(Connelly et al., Chem. Rev. 96:877-910 (1996), incorporated byreference) and electrochemical (Geiger et al., Advances inOrganometallic Chemistry 23:1-93; and Geiger et al., Advances inOrganometallic Chemistry 24:87, incorporated by reference) electrontransfer or “redox” reactions. Metallocene derivatives of a variety ofthe first, second and third row transition metals are potentialcandidates as redox moieties that are covalently attached to either theribose ring or the nucleoside base of nucleic acid. Other potentiallysuitable organometallic ligands include cyclic arenes such as benzene,to yield bis(arene)metal compounds and their ring substituted and ringfused derivatives, of which bis(benzene)chromium is a prototypicalexample, Other acyclic π-bonded ligands such as the allyl(−1) ion, orbutadiene yield potentially suitable organometallic compounds, and allsuch ligands, in conjuction with other π-bonded and δ-bonded ligandsconstitute the general class of organometallic compounds in which thereis a metal to carbon bond. Electrochemical studies of various dimers andoligomers of such compounds with bridging organic ligands, andadditional non-bridging ligands, as well as with and without metal—metalbonds are potential candidate redox moieties in nucleic acid analysis.

When one or more of the co-ligands is an organometallic ligand, theligand is generally attached via one of the carbon atoms of theorganometallic ligand, although attachment may be via other atoms forheterocyclic ligands. Preferred organometallic ligands includemetallocene ligands, including substituted derivatives and themetalloceneophanes (see page 1174 of Cotton and Wilkenson, supra). Forexample, derivatives of metallocene ligands such asmethylcyclopentadienyl, with multiple methyl groups being preferred,such as pentamethylcyclopentadienyl, can be used to increase thestability of the metallocene. In a preferred embodiment, only one of thetwo metallocene ligands of a metallocene are derivatized.

As described herein, any combination of ligands may be used. Preferredcombinations include: a) all ligands are nitrogen donating ligands; b)all ligands are organometallic ligands; and c) the ligand at theterminus of the conductive oligomer is a metallocene ligand and theligand provided by the nucleic acid is a nitrogen donating ligand, withthe other ligands, if needed, are either nitrogen donating ligands ormetallocene ligands, or a mixture. These combinations are depicted inrepresentative structures using the conductive oligomer of Structure 3are depicted in Structures 27 (using phenanthroline and amino asrepresentative ligands), 28 (using ferrocene as the metal-ligandcombination) and 29 (using cyclopentadienyl and amino as representativeligands).

In a preferred embodiment, the ligands used in the invention showaltered fluorescent properties depending on the redox state of thechelated metal ion. As described below, this thus serves as anadditional mode of detection of electron transfer between the ETM andthe electrode.

In addition, similar methods can be used to attach proteins to thedetection electrode; see for example U.S. Pat. No. 5,620,850, herebyincorporated by reference.

In a preferred embodiment, as is described more fully below, the ligandattached to the nucleic acid is an amino group attached to the 2′ or 3′position of a ribose of the ribose-phosphate backbone. This ligand maycontain a multiplicity of amino groups so as to form a polydentateligand which binds the metal ion. Other preferred ligands includecyclopentadiene and phenanthroline.

The use of metal ions to connect the nucleic acids can serve as aninternal control or calibration of the system, to evaluate the number ofavailable nucleic acids on the surface. However, as will be appreciatedby those in the art, if metal ions are used to connect the nucleic acidsto the conductive oligomers, it is generally desirable to have thismetal ion complex have a different redox potential than that of the ETMsused in the rest of the system, as described below. This is generallytrue so as to be able to distinguish the presence of the capture probefrom the presence of the target sequence. This may be useful foridentification, calibration and/or quantification. Thus, the amount ofcapture probe on an electrode may be compared to the amount ofhybridized double stranded nucleic acid to quantify the amount of targetsequence in a sample. This is quite significant to serve as an internalcontrol of the sensor or system. This allows a measurement either priorto the addition of target or after, on the same molecules that will beused for detection, rather than rely on a similar but different controlsystem. Thus, the actual molecules that will be used for the detectioncan be quantified prior to any experiment. This is a significantadvantage over prior methods.

In a preferred embodiment, the capture probe nucleic acids (or otherbinding ligands) are covalently attached to the electrode via aninsulator. The attachment of nucleic acids (and other binding ligands)to insulators such as alkyl groups is well known, and can be done to thebase or the backbone, including the ribose or phosphate for backbonescontaining these moieties, or to alternate backbones for nucleic acidanalogs.

In a preferred embodiment, there may be one or more different captureprobe species on the surface. In some embodiments, there may be one typeof capture probe, or one type of capture probe extender, as is morefully described below. Alternatively, different capture probes, or onecapture probes with a multiplicity of different capture extender probescan be used. Similarly, it may be desirable (particular in the case ofnucleic acid analytes and binding ligands in mechanism-2 systems) to useauxiliary capture probes that comprise relatively short probe sequences,that can be used to “tack down” components of the system, for examplethe recruitment linkers, to increase the concentration of ETMs at thesurface.

Thus the present invention provides substrates comprising at least onedetection electrode comprising monolayers and capture binding ligands,useful in target analyte detection systems.

In a preferred embodiment, the compositions further comprise a solutionor soluble binding ligand, although as is more fully described below,for mechanism-1 systems, the ETMs may be added in the form ofnon-covalently attached hybridization indicators. Solution bindingligands are similar to capture binding ligands, in that they bind,preferably specifically, to target analytes. The solution binding ligandmay be the same or different from the capture binding ligand. Generally,the solution binding ligands are not directed attached to the surface,although as depicted in FIG. 5A they may be. The solution binding ligandeither directly comprises a recruitment linker that comprises at leastone ETM (FIG. 4A), or the recruitment linker binds, either directly(FIG. 4A) or indirectly (FIG. 4E), to the solution binding ligand.

Thus, “solution binding ligands” or “soluble binding ligands” or “signalcarriers” or “label probes” or “label binding ligands” with recruitmentlinkers comprising covalently attached ETMs are provided. That is, oneportion of the label probe or solution binding ligand directly orindirectly binds to the target analyte, and one portion comprises arecruitment linker comprising covalently attached ETMs. In some systems,for example in mechanism-1 nucleic acid systems, these may be the same.Similarly, for mechanism-1 systems, the recruitment linker comprisesnucleic acid that will hybridize to detection probes. The terms“electron donor moiety”, “electron acceptor moiety”, and “ETMs” (ETMs)or grammatical equivalents herein refers to molecules capable ofelectron transfer under certain conditions. It is to be understood thatelectron donor and acceptor capabilities are relative; that is, amolecule which can lose an electron under certain experimentalconditions will be able to accept an electron under differentexperimental conditions. It is to be understood that the number ofpossible electron donor moieties and electron acceptor moieties is verylarge, and that one skilled in the art of electron transfer compoundswill be able to utilize a number of compounds in the present invention.Preferred ETMs include, but are not limited to, transition metalcomplexes, organic ETMs, and electrodes.

In a preferred embodiment, the ETMs are transition metal complexes.Transition metals are those whose atoms have a partial or complete dshell of electrons. Suitable transition metals for use in the inventionare listed above.

The transition metals are complexed with a variety of ligands, L,defined above, to form suitable transition metal complexes, as is wellknown in the art.

In addition to transition metal complexes, other organic electron donorsand acceptors may be covalently attached to the nucleic acid for use inthe invention. These organic molecules include, but are not limited to,riboflavin, xanthene dyes, azine dyes, acridine orange,N,N′-dimethyl-2,7-diazapyrenium dichloride (DAP²⁺), methylviologen,ethidium bromide, quinones such asN,N′-dimethylanthra(2,1,9-def.6,5,10-d′e′f)diisoquinoline dichloride(ADIQ²⁺); porphyrins ([meso-tetrakis(N-methyl-x-pyridinium)porphyrintetrachloride], varlamine blue B hydrochloride, Bindschedler's green;2,6-dichloroindophenol, 2,6-dibromophenolindophenol; Brilliant crestblue (3-amino-9-dimethyl-amino-10-methylphenoxyazine chloride),methylene blue; Nile blue A (aminoaphthodiethylaminophenoxazinesulfate), indigo-5,5′,7,7′-tetrasulfonic acid, indigo-5,5′,7-trisulfonicacid; phenosafranine, indigo-5-monosulfonic acid; safranine T;bis(dimethylglyoximato)-iron(II) chloride; induline scarlet, neutralred, anthracene, coronene, pyrene, 9-phenylanthracene, rubrene,binaphthyl, DPA, phenothiazene, fluoranthene, phenanthrene, chrysene,1,8-diphenyl-1,3,5,7-octatetracene, naphthalene, acenaphthalene,perylene, TMPD and analogs and subsitituted derivatives of thesecompounds.

In one embodiment, the electron donors and acceptors are redox proteinsas are known in the art. However, redox proteins in many embodiments arenot preferred.

In one embodiment, particularly when an electrophoresis step is used,the ETMs are chosen to be charged molecules, preferably when the targetanalyte is not charged. Thus, for example, solution binding ligands thateither directly or indirectly contain a number of charged ETMs can bebound to the target analyte prior to electrophoresis, to allow thetarget analyte to have a sufficient charge to move within the electricfield, thus providing a dual purpose of providing charge and a detectionmoiety. Thus for example, label probes that contain charged ETMs may beused, that bind either directly to the target analyte or to anintermediate species such as an amplifier probe can be used.Alternatively, other charged species can be added in addition to theETMs. Alternatively, these charges species may also be an integral partof the system; for example, part of the label probe may be a chargedpolymer such as polylysine. However, in this embodiment, the migrationof non-specifically bound label probes to the detection surface canresult in an increase in non-specific signals. Therefore, in thisembodiment, the use of a reverse electric field (generally a pulse ofreverse polarity) after electrophoresis can result in thenon-specifically bound label probes being driven off or away from thedetection probe surface, to decrease the background non-specific signal.

The choice of the specific ETMs will be influenced by the type ofelectron transfer detection used, as is generally outlined below.Preferred ETMs are metallocenes, with ferrocene, including derivatives,being particularly preferred.

In a preferred embodiment, a plurality of ETMs are used. As is shown inthe examples, the use of multiple ETMs provides signal amplification andthus allows more sensitive detection limits. As discussed below, whilethe use of multiple ETMs on nucleic acids that hybridize tocomplementary strands can cause decreases in T_(m)s of the hybridizationcomplexes depending on the number, site of attachment and spacingbetween the multiple ETMs, this is not a factor when the ETMs are on therecruitment linker (i.e. “mechanism-2” systems), since this does nothybridize to a complementary sequence. Accordingly, pluralities of ETMsare preferred, with at least about 2 ETMs per recruitment linker beingpreferred, and at least about 10 being particularly preferred, and atleast about 20 to 50 being especially preferred. In some instances, verylarge numbers of ETMs (50 to 1000) can be used.

Thus, solution binding ligands, or label probes, with covalentlyattached ETMs are provided. The method of attachment of the ETM to thesolution binding ligand will vary depending on the mode of detection(i.e. mechanism-1 or -2 systems) and the composition of the solutionbinding ligand. As is more fully outlined below, in mechanism-2 systems,the portion of the solution binding ligand (or label probe) thatcomprises the ETM is referred to as a “recruitment linker” and cancomprise either nucleic acid or non-nucleic acid. For mechanism-1systems, the recruitment linker must be nucleic acid.

Thus, as will be appreciated by those in the art, there are a variety ofconfigurations that can be used. In a preferred embodiment, therecruitment linker is nucleic acid (including analogs), and attachmentof the ETMs can be via (1) a base; (2) the backbone, including theribose, the phosphate, or comparable structures in nucleic acid analogs;(3) nucleoside replacement, described below; or (4) metallocenepolymers, as described below. In a preferred embodiment, the recruitmentlinker is non-nucleic acid, and can be either a metallocene polymer oran alkyl-type polymer (including heteroalkyl, as is more fully describedbelow) containing ETM substitution groups. These options are generallydepicted in FIG. 7.

In a preferred embodiment, the recruitment linker is a nucleic acid, andcomprises covalently attached ETMs. The ETMs may be attached tonucleosides within the nucleic acid in a variety of positions. Preferredembodiments include, but are not limited to, (1) attachment to the baseof the nucleoside, (2) attachment of the ETM as a base replacement, (3)attachment to the backbone of the nucleic acid, including either to aribose of the ribose-phosphate backbone or to a phosphate moiety, or toanalogous structures in nucleic acid analogs, and (4) attachment viametallocene polymers.

In addition, as is described below, when the recruitment linker isnucleic acid, it may be desirable to use secondary label probes, thathave a first portion that will hybridize to a portion of the primarylabel probes and a second portion comprising a recruitment linker as isdefined herein. This is generally depicted in FIGS. 6Q and 6R; this issimilar to the use of an amplifier probe, except that both the primaryand the secondary label probes comprise ETMs.

In a preferred embodiment, the ETM is attached to the base of anucleoside as is generally outlined above for attachment of theconductive oligomer. Attachment can be to an internal nucleoside or aterminal nucleoside.

The covalent attachment to the base will depend in part on the ETMchosen, but in general is similar to the attachment of conductiveoligomers to bases, as outlined above. Attachment may generally be doneto any position of the base. In a preferred embodiment, the ETM is atransition metal complex, and thus attachment of a suitable metal ligandto the base leads to the covalent attachment of the ETM. Alternatively,similar types of linkages may be used for the attachment of organicETMs, as will be appreciated by those in the art.

In one embodiment, the C4 attached amino group of cytosine, the C6attached amino group of adenine, or the C2 attached amino group ofguanine may be used as a transition metal ligand.

Ligands containing aromatic groups can be attached via acetylenelinkages as is known in the art (see Comprehensive Organic Synthesis,Trost et al., Ed., Pergamon Press, Chapter 2.4: Coupling ReactionsBetween sp² and sp Carbon Centers, Sonogashira, pp521-549, andpp950-953, hereby incorporated by reference). Structure 30 depicts arepresentative structure in the presence of the metal ion and any othernecessary ligands; Structure 30 depicts uridine, although as for all thestructures herein, any other base may also be used.

L_(a) is a ligand, which may include nitrogen, oxygen, sulfur orphosphorus donating ligands or organometallic ligands such asmetallocene ligands. Suitable L_(a) ligands include, but not limited to,phenanthroline, imidazole, bpy and terpy. L_(r) and M are as definedabove. Again, it will be appreciated by those in the art, a linker (“Z”)may be included between the nucleoside and the ETM.

Similarly, as for the conductive oligomers, the linkage may be doneusing a linker, which may utilize an amide linkage (see generally Telseret al., J. Am. Chem. Soc. 111:7221-7226 (1989); Telser et al., J. Am.Chem. Soc. 111:7226-7232 (1989), both of which are expresslyincorporated by reference). These structures are generally depictedbelow in Structure 31, which again uses uridine as the base, although asabove, the other bases may also be used:

In this embodiment, L is a ligand as defined above, with L_(r) and M asdefined above as well. Preferably, L is amino, phen, byp and terpy.

In a preferred embodiment, the ETM attached to a nucleoside is ametallocene; i.e. the L and L_(r) of Structure 31 are both metalloceneligands, L_(m), as described above. Structure 32 depicts a preferredembodiment wherein the metallocene is ferrocene, and the base isuridine, although other bases may be used:

Preliminary data suggest that Structure 32 may cyclize, with the secondacetylene carbon atom attacking the carbonyl oxygen, forming afuran-like structure. Preferred metallocenes include ferrocene,cobaltocene and osmiumocene.

In a preferred embodiment, the ETM is attached to a ribose at anyposition of the ribose-phosphate backbone of the nucleic acid, i.e.either the 5′ or 3′ terminus or any internal nucleoside. Ribose in thiscase can include ribose analogs. As is known in the art, nucleosidesthat are modified at either the 2′ or 3′ position of the ribose can bemade, with nitrogen, oxygen, sulfur and phosphorus-containingmodifications possible. Amino-modified and oxygen-modified ribose ispreferred. See generally PCT publication WO 95/15971, incorporatedherein by reference. These modification groups may be used as atransition metal ligand, or as a chemically functional moiety forattachment of other transition metal ligands and organometallic ligands,or organic electron donor moieties as will be appreciated by those inthe art. In this embodiment, a linker such as depicted herein for “Z”may be used as well, or a conductive oligomer between the ribose and theETM. Preferred embodiments utilize attachment at the 2′ or 3′ positionof the ribose, with the 2′ position being preferred. Thus for example,the conductive oligomers depicted in Structure 13, 14 and 15 may bereplaced by ETMs; alternatively, the ETMs may be added to the freeterminus of the conductive oligomer.

In a preferred embodiment, a metallocene serves as the ETM, and isattached via an amide bond as depicted below in Structure 33. Theexamples outline the synthesis of a preferred compound when themetallocene is ferrocene.

In a preferred embodiment, amine linkages are used, as is generallydepicted in Structure 34.

Z is a linker, as defined herein, with 1-16 atoms being preferred, and2-4 atoms being particularly preferred, and t is either one or zero.

In a preferred embodiment, oxo linkages are used, as is generallydepicted in Structure 35.

In Structure 35, Z is a linker, as defined herein, and t is either oneor zero. Preferred Z linkers include alkyl groups including heteroalkylgroups such as (CH₂)n and (CH₂CH₂O)n, with n from 1 to 10 beingpreferred, and n=1 to 4 being especially preferred, and n=4 beingparticularly preferred.

Linkages utilizing other heteroatoms are also possible.

In a preferred embodiment, an ETM is attached to a phosphate at anyposition of the ribose-phosphate backbone of the nucleic acid. This maybe done in a variety of ways. In one embodiment, phosphodiester bondanalogs such as phosphoramide or phosphoramidite linkages may beincorporated into a nucleic acid, where the heteroatom (i.e. nitrogen)serves as a transition metal ligand (see PCT publication WO 95/15971,incorporated by reference). Alternatively, the conductive oligomersdepicted in Structures 23 and 24 may be replaced by ETMs. In a preferredembodiment, the composition has the structure shown in Structure 36.

In Structure 36, the ETM is attached via a phosphate linkage, generallythrough the use of a linker, Z. Preferred Z linkers include alkylgroups, including heteroalkyl groups such as (CH₂)_(n), (CH₂CH₂O)_(n),with n from 1 to 10 being preferred, and n =1 to 4 being especiallypreferred, and n=4 being particularly preferred.

In mechanism-2 systems, when the ETM is attached to the base or thebackbone of the nucleoside, it is possible to attach the ETMs via“dendrimer” structures, as is more fully outlined below. As is generallydepicted in FIG. 8, alkyl-based linkers can be used to create multiplebranching structures comprising one or more ETMs at the terminus of eachbranch. Generally, this is done by creating branch points containingmultiple hydroxy groups, which optionally can then be used to addadditional branch points. The terminal hydroxy groups can then be usedin phosphoramidite reactions to add ETMs, as is generally done below forthe nucleoside replacement and metallocene polymer reactions.

In a preferred embodiment, an ETM such as a metallocene is used as a“nucleoside replacement”, serving as an ETM. For example, the distancebetween the two cyclopentadiene rings of ferrocene is similar to theorthongonal distance between two bases in a double stranded nucleicacid. Other metallocenes in addition to ferrocene may be used, forexample, air stable metallocenes such as those containing cobalt orruthenium. Thus, metallocene moieties may be incorporated into thebackbone of a nucleic acid, as is generally depicted in Structure 37(nucleic acid with a ribose-phosphate backbone) and Structure 38(peptide nucleic acid backbone). Structures 37 and 38 depict ferrocene,although as will be appreciated by those in the art, other metallocenesmay be used as well. In general, air stable metallocenes are preferred,including metallocenes utilizing ruthenium and cobalt as the metal.

In Structure 37, Z is a linker as defined above, with generally short,alkyl groups, including heteroatoms such as oxygen being preferred.Generally, what is important is the length of the linker, such thatminimal perturbations of a double stranded nucleic acid is effected, asis more fully described below. Thus, methylene, ethylene, ethyleneglycols, propylene and butylene are all preferred, with ethylene andethylene glycol being particularly preferred. In addition, each Z linkermay be the same or different. Structure 37 depicts a ribose-phosphatebackbone, although as will be appreciated by those in the art, nucleicacid analogs may also be used, including ribose analogs and phosphatebond analogs.

In Structure 38, preferred Z groups are as listed above, and again, eachZ linker can be the same or different. As above, other nucleic acidanalogs may be used as well.

In addition, although the structures and discussion above depictsmetallocenes, and particularly ferrocene, this same general idea can beused to add ETMs in addition to metallocenes, as nucleoside replacementsor in polymer embodiments, described below. Thus, for example, when theETM is a transition metal complex other than a metallocene, comprisingone, two or three (or more) ligands, the ligands can be functionalizedas depicted for the ferrocene to allow the addition of phosphoramiditegroups. Particularly preferred in this embodiment are complexescomprising at least two ring (for example, aryl and substituted aryl)ligands, where each of the ligands comprises functional groups forattachment via phosphoramidite chemistry. As will be appreciated bythose in the art, this type of reaction, creating polymers of ETMseither as a portion of the backbone of the nucleic acid or as “sidegroups” of the nucleic acids, to allow amplification of the signalsgenerated herein, can be done with virtually any ETM that can befunctionalized to contain the correct chemical groups.

Thus, by inserting a metallocene such as ferrocene (or other ETM) intothe backbone of a nucleic acid, nucleic acid analogs are made; that is,the invention provides nucleic acids having a backbone comprising atleast one metallocene. This is distinguished from nucleic acids havingmetallocenes attached to the backbone, i.e. via a ribose, a phosphate,etc. That is, two nucleic acids each made up of a traditional nucleicacid or analog (nucleic acids in this case including a singlenucleoside), may be covalently attached to each other via a metallocene.Viewed differently, a metallocene derivative or substituted metalloceneis provided, wherein each of the two aromatic rings of the metallocenehas a nucleic acid substitutent group.

In addition, as is more fully outlined below, it is possible toincorporate more than one metallocene into the backbone, either withnucleotides in between and/or with adjacent metallocenes. When adjacentmetallocenes are added to the backbone, this is similar to the processdescribed below as “metallocene polymers”; that is, there are areas ofmetallocene polymers within the backbone.

In addition to the nucleic acid substitutent groups, it is alsodesirable in some instances to add additional substituent groups to oneor both of the aromatic rings of the metallocene (or ETM). For example,as these nucleoside replacements are generally part of probe sequencesto be hybridized with a substantially complementary nucleic acid, forexample a target sequence or another probe sequence, it is possible toadd substitutent groups to the metallocene rings to facilitate hydrogenbonding to the base or bases on the opposite strand. These may be addedto any position on the metallocene rings. Suitable substitutent groupsinclude, but are not limited to, amide groups, amine groups, carboxylicacids, and alcohols, including substituted alcohols. In addition, thesesubstitutent groups can be attached via linkers as well, although ingeneral this is not preferred.

In addition, substituent groups on an ETM, particularly metallocenessuch as ferrocene, may be added to alter the redox properties of theETM. Thus, for example, in some embodiments, as is more fully describedbelow, it may be desirable to have different ETMs attached in differentways (i.e. base or ribose attachment), on different probes, or fordifferent purposes (for example, calibration or as an internalstandard). Thus, the addition of substituent groups on the metallocenemay allow two different ETMs to be distinguished.

In order to generate these metallocene-backbone nucleic acid analogs,the intermediate components are also provided. Thus, in a preferredembodiment, the invention provides phosphoramidite metallocenes, asgenerally depicted in Structure 39:

In Structure 39, PG is a protecting group, generally suitable for use innucleic acid synthesis, with DMT, MMT and TMT all being preferred. Thearomatic rings can either be the rings of the metallocene, or aromaticrings of ligands for transition metal complexes or other organic ETMs.The aromatic rings may be the same or different, and may be substitutedas discussed herein. Structure 40 depicts the ferrocene derivative:

These phosphoramidite analogs can be added to standard oligonucleotidesyntheses as is known in the art.

Structure 41 depicts the ferrocene peptide nucleic acid (PNA) monomer,that can be added to PNA synthesis as is known in the art:

In Structure 41, the PG protecting group is suitable for use in peptidenucleic acid synthesis, with MMT, boc and Fmoc being preferred.

These same intermediate compounds can be used to form ETM or metallocenepolymers, which are added to the nucleic acids, rather than as backbonereplacements, as is more fully described below. In a preferredembodiment, particularly for use in mechanism-2 systems, the ETMs areattached as polymers, for example as metallocene polymers, in a“branched” configuration similar to the “branched DNA” embodimentsherein and as outlined in U.S. Pat. No. 5,124,246, using modifiedfunctionalized nucleotides. The general idea is as follows. A modifiedphosphoramidite nucleotide is generated that can ultimately contain afree hydroxy group that can be used in the attachment of phosphoramiditeETMs such as metallocenes. This free hydroxy group could be on the baseor the backbone, such as the ribose or the phosphate (although as willbe appreciated by those in the art, nucleic acid analogs containingother structures can also be used). The modified nucleotide isincorporated into a nucleic acid, and any hydroxy protecting groups areremoved, thus leaving the free hydroxyl. Upon the addition of aphosphoramidite ETM such as a metallocene, as described above instructures 39 and 40, ETMs, such as metallocene ETMs, are added.Additional phosphoramidite ETMs such as metallocenes can be added, toform “ETM polymers”, including “metallocene polymers” as depicted inFIG. 9 with ferrocene. In addition, in some embodiments, it is desirableto increase the solubility of the polymers by adding a “capping” groupto the terminal ETM in the polymer, for example a final phosphate groupto the metallocene as is generally depicted in FIG. 9. Other suitablesolubility enhancing “capping” groups will be appreciated by those inthe art. It should be noted that these solubility enhancing groups canbe added to the polymers in other places, including to the ligand rings,for example on the metallocenes as discussed herein

A preferred embodiment of this general idea is outlined in the Figures.In this embodiment, the 2′ position of a ribose of a phosphoramiditenucleotide is first functionalized to contain a protected hydroxy group,in this case via an oxo-linkage, although any number of linkers can beused, as is generally described herein for Z linkers. The protectedmodified nucleotide is then incorporated via standard phosphoramiditechemistry into a growing nucleic acid. The protecting group is removed,and the free hydroxy group is used, again using standard phosphoramiditechemistry to add a phosphoramidite metallocene such as ferrocene. Asimilar reaction is possible for nucleic acid analogs. For example,using peptide nucleic acids and the metallocene monomer shown inStructure 41, peptide nucleic acid structures containing metallocenepolymers could be generated.

Thus, the present invention provides recruitment linkers of nucleicacids comprising “branches” of metallocene polymers as is generallydepicted in FIGS. 8 and 9. Preferred embodiments also utilizemetallocene polymers from one to about 50 metallocenes in length, withfrom about 5 to about 20 being preferred and from about 5 to about 10being especially preferred.

In addition, when the recruitment linker is nucleic acid, anycombination of ETM attachments may be done. In general, as outlinedherein, when mechanism-1 systems are used, clusters of nucleosidescontaining ETMs can decrease the Tm of hybridization of the probe to itstarget sequence; thus in general, for mechanism-1 systems, the ETMs arespaced out over the length of the sequence, or only small numbers ofthem are used.

In mechanism-1 systems, non-covalently attached ETMs may be used. In oneembodiment, the ETM is a hybridization indicator. Hybridizationindicators serve as an ETM that will preferentially associate withdouble stranded nucleic acid is added, usually reversibly, similar tothe method of Millan et al., Anal. Chem. 65:2317-2323 (1993); Millan etal., Anal. Chem. 662943-2948 (1994), both of which are hereby expresslyincorporated by reference. In this embodiment, increases in the localconcentration of ETMs, due to the association of the ETM hybridizationindicator with double stranded nucleic acid at the surface, can bemonitored using the monolayers comprising the conductive oligomers.Hybridization indicators include intercalators and minor and/or majorgroove binding moieties. In a preferred embodiment, intercalators may beused; since intercalation generally only occurs in the presence ofdouble stranded nucleic acid, only in the presence of double strandednucleic acid will the ETMs concentrate. Intercalating transition metalcomplex ETMs are known in the art. Similarly, major or minor groovebinding moieties, such as methylene blue, may also be used in thisembodiment.

In addition, the binding acceleration systems of the invention may beused in virtually any method that relies on electrochemical detection oftarget analytes, with particular utility in nucleic acid detection. Forexample, the methods and compositions of the invention can be used innucleic acid detection methods that rely on the detection of ETMs thatare inherent to the target analyte. For example, as is generallydescribed in Napier et al., Bioconj. Chem. 8:906 (1997), herebyexpressly incorporated by reference, the guanine bases of nucleic acidcan be detected via changes in the redox state, i.e. guanine oxidationby ruthenium complexes. Similarly, the methods of the invention find usein detection systems that utilize copper surfaces as catalyticelectrodes to oxidize the riboses of nucleic acids.

In a preferred embodiment, the recruitment linker is not nucleic acid,and instead may be any sort of linker or polymer. As will be appreciatedby those in the art, generally any linker or polymer that can bemodified to contain ETMs can be used. In general, the polymers orlinkers should be reasonably soluble and contain suitable functionalgroups for the addition of ETMs.

As used herein, a “recruitment polymer” comprises at least two or threesubunits, which are covalently attached. At least some portion of themonomeric subunits contain functional groups for the covalent attachmentof ETMs. In some embodiments coupling moieties are used to covalentlylink the subunits with the ETMs. Preferred functional groups forattachment are amino groups, carboxy groups, oxo groups and thiolgroups, with amino groups being particularly preferred. As will beappreciated by those in the art, a wide variety of recruitment polymersare possible.

Suitable linkers include, but are not limited to, alkyl linkers(including heteroalkyl (including (poly)ethylene glycol-typestructures), substituted alkyl, aryalkyl linkers, etc. As above for thepolymers, the linkers will comprise one or more functional groups forthe attachment of ETMs, which will be done as will be appreciated bythose in the art, for example through the use homo-orhetero-bifunctional linkers as are well known (see 1994 Pierce ChemicalCompany catalog, technical section on cross-linkers, pages 155-200,incorporated herein by reference).

Suitable recruitment polymers include, but are not limited to,functionalized styrenes, such as amino styrene, functionalized dextrans,and polyamino acids. Preferred polymers are polyamino acids (bothpoly-D-amino acids and poly-L-amino acids), such as polylysine, andpolymers containing lysine and other amino acids being particularlypreferred. As outlined above, in some embodiments, charged recruitmentlinkers are preferred, for example when non-charged target analytes areto be detected. Other suitable polyamino acids are polyglutamic acid,polyaspartic acid, co-polymers of lysine and glutamic or aspartic acid,co-polymers of lysine with alanine, tyrosine, phenylalanine, serine,tryptophan, and/or proline.

In a preferred embodiment, the recruitment linker comprises ametallocene polymer, as is described above.

The attachment of the recruitment linkers to the first portion of thelabel probe, i.e. the portion that binds either directly or indirectlyto the target analyte, will depend on the composition of the recruitmentlinker, as will be appreciated by those in the art. When the recruitmentlinker is nucleic acid, it is generally formed during the synthesis ofthe first portion of the label probe, with incorporation of nucleosidescontaining ETMs as required. Alternatively, the first portion of thelabel probe and the recruitment linker may be made separately, and thenattached. For example, there may be an overlapping section ofcomplementarity, forming a section of double stranded nucleic acid thatcan then be chemically crosslinked, for example by using psoralen as isknown in the art.

When non-nucleic acid recruitment linkers are used, attachment of thelinker/polymer of the recruitment linker will be done generally usingstandard chemical techniques, such as will be appreciated by those inthe art. For example, when alkyl-based linkers are used, attachment canbe similar to the attachment of insulators to nucleic acids.

In addition, it is possible to have recruitment linkers that aremixtures of nucleic acids and non-nucleic acids, either in a linear form(i.e. nucleic acid segments linked together with alkyl linkers) or inbranched forms (nucleic acids with alkyl “branches” that may containETMs and may be additionally branched).

In a preferred embodiment, for example when the target analyte is anucleic acid, it is the target sequence itself that carries the ETMs,rather than the recruitment linker of a label probe. For example, as ismore fully described below, it is possible to enzymatically addtriphosphate nucleotides comprising the ETMs of the invention to agrowing nucleic acid, for example during a polymerase chain reaction(PCR). As will be recognized by those in the art, while several enzymeshave been shown to generally tolerate modified nucleotides, some of themodified nucleotides of the invention, for example the “nucleosidereplacement” embodiments and putatively some of the phosphateattachments, may or may not be recognized by the enzymes to allowincorporation into a growing nucleic acid. Therefore, preferredattachments in this embodiment are to the base or ribose of thenucleotide.

Thus, for example, PCR amplification of a target sequence, as is wellknown in the art, will result in target sequences comprising ETMs,generally randomly incorporated into the sequence. The system of theinvention can then be configured to allow detection using these ETMs, asis generally depicted in FIGS. 6A and 6B.

Alternatively, as outlined more fully below, it is possible toenzymatically add nucleotides comprising ETMs to the terminus of anucleic acid, for example a target nucleic acid. In this embodiment, aneffective “recruitment linker” is added to the terminus of the targetsequence, that can then be used for detection, as is generally depictedin FIG. 6Q. Thus the invention provides compositions utilizingelectrodes comprising monolayers of conductive oligomers and captureprobes, and target sequences that comprises a first portion that iscapable of hybridizing to a component of an assay complex, and a secondportion that does not hybridize to a component of an assay complex andcomprises at least one covalently attached electron transfer moiety.Similarly, methods utilizing these compositions are also provided.

It is also possible to have ETMs connected to probe sequences, i.e.sequences designed to hybridize to complementary sequences, i.e. inmechanism-1 sequences, although this may also be used in mechanism-2systems. Thus, ETMs may be added to non-recruitment linkers as well. Forexample, there may be ETMs added to sections of label probes that dohybridize to components of the assay complex, for example the firstportion, or to the target sequence as outlined above and depicted inFIG. 6R. These ETMs may be used for electron transfer detection in someembodiments, or they may not, depending on the location and system. Forexample, in some embodiments, when for example the target sequencecontaining randomly incorporated ETMs is hybridized directly to thecapture probe, as is depicted in FIGS. 6A and 6B, there may be ETMs inthe portion hybridizing to the capture probe. If the capture probe isattached to the electrode using a conductive oligomer, these ETMs can beused to detect electron transfer as has been previously described.Alternatively, these ETMs may not be specifically detected.

Similarly, in some embodiments, when the recruitment linker is nucleicacid, it may be desirable in some instances to have some or all of therecruitment linker be double stranded, for example in the mechanism-2systems. In one embodiment, there may be a second recruitment linker,substantially complementary to the first recruitment linker, that canhybridize to the first recruitment linker. In a preferred embodiment,the first recruitment linker comprises the covalently attached ETMs. Inan alternative embodiment, the second recruitment linker contains theETMs, and the first recruitment linker does not, and the ETMs arerecruited to the surface by hybridization of the second recruitmentlinker to the first. In yet another embodiment, both the first andsecond recruitment linkers comprise ETMs. It should be noted, asdiscussed above, that nucleic acids comprising a large number of ETMsmay not hybridize as well, i.e. the Tm may be decreased, depending onthe site of attachment and the characteristics of the ETM. Thus, ingeneral, when multiple ETMs are used on hybridizing strands, i.e. inmechanism-1 systems, generally there are less than about 5, with lessthan about 3 being preferred, or alternatively the ETMs should be spacedsufficiently far apart that the intervening nucleotides can sufficientlyhybridize to allow good kinetics.

In a preferred embodiment, the compositions of the invention are used todetect target analytes in a sample. In a preferred embodiment, thetarget analyte is a nucleic acid, and target sequences are detected. Theterm “target sequence” or grammatical equivalents herein means a nucleicacid sequence on a single strand of nucleic acid. The target sequencemay be a portion of a gene, a regulatory sequence, genomic DNA, cDNA,RNA including mRNA and rRNA, or others. It may be any length, with theunderstanding that longer sequences are more specific. As will beappreciated by those in the art, the complementary target sequence maytake many forms. For example, it may be contained within a largernucleic acid sequence, i.e. all or part of a gene or mRNA, a restrictionfragment of a plasmid or genomic DNA, among others. As is outlined morefully below, probes are made to hybridize to target sequences todetermine the presence or absence of the target sequence in a sample.Generally speaking, this term will be understood by those skilled in theart. The target sequence may also be comprised of different targetdomains; for example, a first target domain of the sample targetsequence may hybridize to a capture probe or a portion of captureextender probe, a second target domain may hybridize to a portion of anamplifier probe, a label probe, or a different capture or captureextender probe, etc. The target domains may be adjacent or separated.The terms “first” and “second” are not meant to confer an orientation ofthe sequences with respect to the 5′-3′ orientation of the targetsequence. For example, assuming a 5′-3′ orientation of the complementarytarget sequence, the first target domain may be located either 5′ to thesecond domain, or 3′ to the second domain.

If required, the target analyte is prepared using known techniques. Forexample, the sample may be treated to lyse the cells, using known lysisbuffers, electroporation, etc., with purification and/or amplificationsuch as PCR occuring as needed, as will be appreciated by those in theart.

For nucleic acid systems, the probes of the present invention aredesigned to be complementary to a target sequence (either the targetsequence of the sample or to other probe sequences, as is describedbelow), such that hybridization of the target sequence and the probes ofthe present invention occurs. As outlined below, this complementarityneed not be perfect; there may be any number of base pair mismatcheswhich will interfere with hybridization between the target sequence andthe single stranded nucleic acids of the present invention. However, ifthe number of mutations is so great that no hybridization can occurunder even the least stringent of hybridization conditions, the sequenceis not a complementary target sequence. Thus, by “substantiallycomplementary” herein is meant that the probes are sufficientlycomplementary to the target sequences to hybridize under normal reactionconditions.

Generally, the nucleic acid compositions of the invention are useful asoligonucleotide probes. As is appreciated by those in the art, thelength of the probe will vary with the length of the target sequence andthe hybridization and wash conditions. Generally, oligonucleotide probesrange from about 8 to about 50 nucleotides, with from about 10 to about30 being preferred and from about 12 to about 25 being especiallypreferred. In some cases, very long probes may be used, e.g. 50 to200-300 nucleotides in length. Thus, in the structures depicted herein,nucleosides may be replaced with nucleic acids.

A variety of hybridization conditions may be used in the presentinvention, including high, moderate and low stringency conditions; seefor example Maniatis et al., Molecular Cloning: A Laboratory Manual, 2dEdition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, etal, hereby incorporated by reference. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures. Anextensive guide to the hybridization of nucleic acids is found inTijssen, Techniques in Biochemistry and Molecular Biology—Hybridizationwith Nucleic Acid Probes, “Overview of principles of hybridization andthe strategy of nucleic acid assays” (1993). Generally, stringentconditions are selected to be about 5-10° C. lower than the thermalmelting point (Tm) for the specific sequence at a defined ionic strengthpH. The Tm is the temperature (under defined ionic strength, pH andnucleic acid concentration) at which 50% of the probes complementary tothe target hybridize to the target sequence at equilibrium (as thetarget sequences are present in excess, at Tm, 50% of the probes areoccupied at equilibrium). Stringent conditions will be those in whichthe salt concentration is less than about 1.0 sodium ion, typicallyabout 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0to 8.3 and the temperature is at least about 30° C. for short probes(e.g. 10 to 50 nucleotides) and at least about 60° C. for long probes(e.g. greater than 50 nucleotides). Stringent conditions may also beachieved with the addition of destabilizing agents such as formamide.

In another embodiment, less stringent hybridization conditions are used;for example, moderate or low stringency conditions may be used, as areknown in the art; see Maniatis and Ausubel, supra, and Tijssen, supra.

The hybridization conditions may also vary when a non-ionic backbone,i.e. PNA is used, as is known in the art. In addition, cross-linkingagents may be added after target binding to cross-link, i.e. covalentlyattach, the two strands of the hybridization complex.

As will be appreciated by those in the art, the systems of the inventionmay take on a large number of different configurations, as is generallydepicted in FIGS. 3, 4, 5 and 6. In general, there are three types ofsystems that can be used: (1) systems in which the target sequenceitself is labeled with ETMs (see FIGS. 4A, 5A, 5B and 5D; this isgenerally useful for nucleic acid systems); (2) systems in which labelprobes directly bind to the target analytes (see FIGS. 4C and 4H fornucleic acid examples and FIGS. 6A, 6B, 6D and 6E, for examples ofnon-nucleic acid analytes); and (3) systems in which label probes areindirectly bound to the target sequences, for example through the use ofamplifier probes (see FIGS. 4C, 5E, 5F and 5G for nucleic acid examplesand FIG. 6C for representative non-nucleic acid target analytes).

In all three of these systems, it is preferred, although not required,that the target sequence be immobilized on the electrode surface. Thisis preferably done using capture probes and optionally one or morecapture extender probes; see FIG. 3 for representative nucleic acidexamples. When only capture probes are utilized, it is necessary to haveunique capture probes for each target sequence; that is, the surfacemust be customized to contain unique capture probes. Alternatively,capture extender probes may be used, that allow a “universal” surface,i.e. a surface containing a single type of capture probe that can beused to detect any target sequence. “Capture extender” probes aregenerally depicted in FIGS. 4C, 5C, 5E, 5G and 5H, as well as FIG. 6B,etc., and have a first portion that will hybridize to all or part of thecapture probe, and a second portion that will hybridize to a portion ofthe target sequence. This then allows the generation of customizedsoluble probes, which as will be appreciated by those in the art isgenerally simpler and less costly. As shown herein, two capture extenderprobes may be used. This has generally been done to stabilize assaycomplexes (for example when the target sequence is large, or when largeamplifier probes (particularly branched or dendrimer amplifier probes)are used.

While the discussion and figures herein generally depict nucleic acidembodiments, these same ideas can be used for non-nucleic acid targetanalytes. For example, capture extender ligands can be generated, aswill be appreciated by those in the art. For example, a nucleic acid“tail” can be added to a binding ligand, as is generally depicted inFIG. 6B.

In a preferred embodiment, the binding ligands are added after theformation of the SAM ((4) above). This may be done in a variety of ways,as will be appreciated by those in the art. In one embodiment,conductive oligomers with terminal functional groups are made, withpreferred embodiments utilizing activated carboxylates andisothiocyanates, that will react with primary amines that are eitherpresent or put onto the binding ligand such as a nucleic acid, using anactivated carboxylate. These two reagents have the advantage of beingstable in aqueous solution, yet react with primary alkylamines. However,the primary aromatic amines and secondary and tertiary amines of thebases should not react, thus allowing site specific addition of nucleicacids to the surface. Similar techniques can be used with non-nucleicacid components; for example, as outlined above, the attachment ofproteins to SAMs comprising metal chelates is known; see U.S. Pat. No.5,620,850. This allows the spotting of probes (either capture ordetection probes, or both) using known methods (ink jet, spotting, etc.)onto the surface.

In addition, there are a number of non-nucleic acid methods that can beused to immobilize a nucleic acid on a surface. For example, bindingpartner pairs can be utilized; i.e. one binding partner is attached tothe terminus of the conductive oligomer, and the other to the end of thenucleic acid. This may also be done without using a nucleic acid captureprobe; that is, one binding partner serves as the capture probe and theother is attached to either the target sequence or a capture extenderprobe. That is, either the target sequence comprises the bindingpartner, or a capture extender probe that will hybridize to the targetsequence comprises the binding partner. Suitable binding partner pairsinclude, but are not limited to, hapten pairs such asbiotin/streptavidin; antigens/antibodies; NTA/histidine tags; etc. Ingeneral, smaller binding partners are preferred, such that the electronscan pass from the nucleic acid into the conductive oligomer to allowdetection.

In a preferred embodiment, when the target sequence itself is modifiedto contain a binding partner, the binding partner is attached via amodified nucleotide that can be enzymatically attached to the targetsequence, for example during a PCR target amplification step.Alternatively, the binding partner should be easily attached to thetarget sequence.

Alternatively, a capture extender probe may be utilized that has anucleic acid portion for hybridization to the target as well as abinding partner (for example, the capture extender probe may comprise anon-nucleic acid portion such as an alkyl linker that is used to attacha binding partner). In this embodiment, it may be desirable tocross-link the double-stranded nucleic acid of the target and captureextender probe for stability, for example using psoralen as is known inthe art.

In one embodiment, the target is not bound to the electrode surfaceusing capture probes. In this embodiment, what is important, as for allthe assays herein, is that excess label probes be removed prior todetection and that the assay complex (the recruitment linker) be inproximity to the surface. As will be appreciated by those in the art,this may be accomplished in other ways. For example, the assay complexmay be present on beads that are added to the electrode comprising themonolayer. The recruitment linkers comprising the ETMs may be placed inproximity to the conductive oligomer surface using techniques well knownin the art, including gravity settling of the beads on the surface,electrostatic or magnetic interactions between bead components and thesurface, using binding partner attachment as outlined above.Alternatively, after the removal of excess reagents such as excess labelprobes, the assay complex may be driven down to the surface, for examplevia electrophoresis as is outlined herein.

However, preferred embodiments utilize assay complexes attached vianucleic acid capture probes.

In a preferred embodiment, the target sequence itself contains the ETMs.As discussed above, this may be done using target sequences that haveETMs incorporated at any number of positions, as outlined above. In thisembodiment, as for the others of the system, the 3′-5′ orientation ofthe probes and targets is chosen to get the ETM-containing structures(i.e. recruitment linkers or target sequences) as close to the surfaceof the monolayer as possible, and in the correct orientation. This maybe done using attachment via insulators or conductive oligomers as isgenerally shown in the Figures. In addition, as will be appreciated bythose in the art, multiple capture probes can be utilized, either in aconfiguration such as depicted in FIG. 5D, wherein the 5′-3′ orientationof the capture probes is different, or where “loops” of target form whenmultiples of capture probes are used.

In a preferred embodiment, the label probes directly hybridize to thetarget sequences, as is generally depicted in the figures. In theseembodiments, the target sequence is preferably, but not required to be,immobilized on the surface using capture probes, including captureextender probes. Label probes are then used to bring the ETMs intoproximity of the surface of the monolayer comprising conductiveoligomers. In a preferred embodiment, multiple label probes are used;that is, label probes are designed such that the portion that hybridizesto the target sequence can be different for a number of different labelprobes, such that amplification of the signal occurs, since multiplelabel probes can bind for every target sequence. Thus, as depicted inthe figures, n is an integer of at least one. Depending on thesensitivity desired, the length of the target sequence, the number ofETMs per label probe, etc., preferred ranges of n are from 1 to 50, withfrom about 1 to about 20 being particularly preferred, and from about 2to about 5 being especially preferred. In addition, if “generic” labelprobes are desired, label extender probes can be used as generallydescribed below for use with amplifier probes.

As above, generally in this embodiment the configuration of the systemand the label probes are designed to recruit the ETMs as close aspossible to the monolayer surface.

In a preferred embodiment, the label probes are hybridized to the targetsequence indirectly. That is, the present invention finds use in novelcombinations of signal amplification technologies and electron transferdetection on electrodes, which may be particularly useful in sandwichhybridization assays, as generally depicted in the Figures for nucleicacid embodiments; similar systems can be developed for non-nucleic acidtarget analytes. In these embodiments, the amplifier probes of theinvention are bound to the target sequence in a sample either directlyor indirectly. Since the amplifier probes preferably contain arelatively large number of amplification sequences that are availablefor binding of label probes, the detectable signal is significantlyincreased, and allows the detection limits of the target to besignificantly improved. These label and amplifier probes, and thedetection methods described herein, may be used in essentially any knownnucleic acid hybridization formats, such as those in which the target isbound directly to a solid phase or in sandwich hybridization assays inwhich the target is bound to one or more nucleic acids that are in turnbound to the solid phase.

In general, these embodiments may be described as follows withparticular reference to nucleic acids. An amplifier probe is hybridizedto the target sequence, either directly (e.g. FIGS. 4C and 5E), orthrough the use of a label extender probe (e.g. FIGS. 5F and 5G), whichserves to allow “generic” amplifier probes to be made. The targetsequence is preferably, but not required to be, immobilized on theelectrode using capture probes. Preferably, the amplifier probe containsa multiplicity of amplification sequences, although in some embodiments,as described below, the amplifier probe may contain only a singleamplification sequence. The amplifier probe may take on a number ofdifferent forms; either a branched conformation, a dendrimerconformation, or a linear “string” of amplification sequences. Theseamplification sequences are used to form hybridization complexes withlabel probes, and the ETMs can be detected using the electrode.

Accordingly, the present invention provides assay complexes comprisingat least one amplifier probe. By “amplifier probe” or “nucleic acidmultimer” or “amplification multimer” or grammatical equivalents hereinis meant a nucleic acid probe that is used to facilitate signalamplification. Amplifier probes comprise at least a firstsingle-stranded nucleic acid probe sequence, as defined below, and atleast one single-stranded nucleic acid amplification sequence, with amultiplicity of amplification sequences being preferred.

Amplifier probes comprise a first probe sequence that is used, eitherdirectly or indirectly, to hybridize to the target sequence. That is,the amplifier probe itself may have a first probe sequence that issubstantially complementary to the target sequence (e.g. FIG. 5E), or ithas a first probe sequence that is substantially complementary to aportion of an additional probe, in this case called a label extenderprobe, that has a first portion that is substantially complementary tothe target sequence (e.g. FIGS. 5F and 5G). In a preferred embodiment,the first probe sequence of the amplifier probe is substantiallycomplementary to the target sequence, as is generally depicted in FIG.5E.

In general, as for all the probes herein, the first probe sequence is ofa length sufficient to give specificity and stability. Thus generally,the probe sequences of the invention that are designed to hybridize toanother nucleic acid (i.e. probe sequences, amplification sequences,portions or domains of larger probes) are at least about 5 nucleosideslong, with at least about 10 being preferred and at least about 15 beingespecially preferred.

In a preferred embodiment, the amplifier probes, or any of the otherprobes of the invention, may form hairpin stem-loop structures in theabsence of their target. The length of the stem double-stranded sequencewill be selected such that the hairpin structure is not favored in thepresence of target. The use of these type of probes, in the systems ofthe invention or in any nucleic acid detection systems, can result in asignificant decrease in non-specific binding and thus an increase in thesignal to noise ratio.

Generally, these hairpin structures comprise four components. The firstcomponent is a target binding sequence, i.e. a region complementary tothe target (which may be the sample target sequence or another probesequence to which binding is desired), that is about 10 nucleosideslong, with about 15 being preferred. The second component is a loopsequence, that can facilitate the formation of nucleic acid loops.Particularly preferred in this regard are repeats of GTC, which has beenidentified in Fragile X Syndrome as forming turns. (When PNA analogs areused, turns comprising proline residues may be preferred). Generally,from three to five repeats are used, with four to five being preferred.The third component is a self-complementary region, which has a firstportion that is complementary to a portion of the target sequence regionand a second portion that comprises a first portion of the label probebinding sequence. The fourth component is substantially complementary toa label probe (or other probe, as the case may be). The fourth componentfurther comprises a “sticky end”, that is, a portion that does nothybridize to any other portion of the probe, and preferably containsmost, if not all, of the ETMs. As will be appreciated by those in theart, the any or all of the probes described herein may be configured toform hairpins in the absence of their targets, including the amplifier,capture, capture extender, label and label extender probes.

In a preferred embodiment, several different amplifier probes are used,each with first probe sequences that will hybridize to a differentportion of the target sequence. That is, there is more than one level ofamplification; the amplifier probe provides an amplification of signaldue to a multiplicity of labelling events, and several differentamplifier probes, each with this multiplicity of labels, for each targetsequence is used. Thus, preferred embodiments utilize at least twodifferent pools of amplifier probes, each pool having a different probesequence for hybridization to different portions of the target sequence;the only real limitation on the number of different amplifier probeswill be the length of the original target sequence. In addition, it isalso possible that the different amplifier probes contain differentamplification sequences, although this is generally not preferred.

In a preferred embodiment, the amplifier probe does not hybridize to thesample target sequence directly, but instead hybridizes to a firstportion of a label extender probe, as is generally depicted in FIG. 5F.This is particularly useful to allow the use of “generic” amplifierprobes, that is, amplifier probes that can be used with a variety ofdifferent targets. This may be desirable since several of the amplifierprobes require special synthesis techniques. Thus, the addition of arelatively short probe as a label extender probe is preferred. Thus, thefirst probe sequence of the amplifier probe is substantiallycomplementary to a first portion or domain of a first label extendersingle-stranded nucleic acid probe. The label extender probe alsocontains a second portion or domain that is substantially complementaryto a portion of the target sequence. Both of these portions arepreferably at least about 10 to about 50 nucleotides in length, with arange of about 15 to about 30 being preferred. The terms “first” and“second” are not meant to confer an orientation of the sequences withrespect to the 5′-3′ orientation of the target or probe sequences. Forexample, assuming a 5′-3′ orientation of the complementary targetsequence, the first portion may be located either 5′ to the secondportion, or 3′ to the second portion. For convenience herein, the orderof probe sequences are generally shown from left to right.

In a preferred embodiment, more than one label extender probe-amplifierprobe pair may be used, tht is, n is more than 1. That is, a pluralityof label extender probes may be used, each with a portion that issubstantially complementary to a different portion of the targetsequence; this can serve as another level of amplification. Thus, apreferred embodiment utilizes pools of at least two label extenderprobes, with the upper limit being set by the length of the targetsequence.

In a preferred embodiment, more than one label extender probe is usedwith a single amplifier probe to reduce non-specific binding, as isdepicted in FIG. 5G and generally outlined in U.S. Pat. No. 5,681,697,incorporated by reference herein. In this embodiment, a first portion ofthe first label extender probe hybridizes to a first portion of thetarget sequence, and the second portion of the first label extenderprobe hybridizes to a first probe sequence of the amplifier probe. Afirst portion of the second label extender probe hybridizes to a secondportion of the target sequence, and the second portion of the secondlabel extender probe hybridizes to a second probe sequence of theamplifier probe. These form structures sometimes referred to as“cruciform” structures or configurations, and are generally done toconfer stability when large branched or dendrimeric amplifier probes areused.

In addition, as will be appreciated by those in the art, the labelextender probes may interact with a preamplifier probe, described below,rather than the amplifier probe directly.

Similarly, as outlined above, a preferred embodiment utilizes severaldifferent amplifier probes, each with first probe sequences that willhybridize to a different portion of the label extender probe. Inaddition, as outlined above, it is also possible that the differentamplifier probes contain different amplification sequences, althoughthis is generally not preferred.

In addition to the first probe sequence, the amplifier probe alsocomprises at least one amplification sequence. An “amplificationsequence” or “amplification segment” or grammatical equivalents hereinis meant a sequence that is used, either directly or indirectly, to bindto a first portion of a label probe as is more fully described below.Preferably, the amplifier probe comprises a multiplicity ofamplification sequences, with from about 3 to about 1000 beingpreferred, from about 10 to about 100 being particularly preferred, andabout 50 being especially preferred. In some cases, for example whenlinear amplifier probes are used, from 1 to about 20 is preferred withfrom about 5 to about 10 being particularly preferred.

The amplification sequences may be linked to each other in a variety ofways, as will be appreciated by those in the art. They may be covalentlylinked directly to each other, or to intervening sequences or chemicalmoieties, through nucleic acid linkages such as phosphodiester bonds,PNA bonds, etc., or through interposed linking agents such amino acid,carbohydrate or polyol bridges, or through other cross-linking agents orbinding partners. The site(s) of linkage may be at the ends of asegment, and/or at one or more internal nucleotides in the strand. In apreferred embodiment, the amplification sequences are attached vianucleic acid linkages.

In a preferred embodiment, branched amplifier probes are used, as aregenerally described in U.S. Pat. No. 5,124,246, hereby incorporated byreference. Branched amplifier probes may take on “fork-like” or“comb-like” conformations. “Fork-like” branched amplifier probesgenerally have three or more oligonucleotide segments emanating from apoint of origin to form a branched structure. The point of origin may beanother nucleotide segment or a multifunctional molecule to whcih atleast three segments can be covalently or tightly bound. “Comb-like”branched amplifier probes have a linear backbone with a multiplicity ofsidechain oligonucleotides extending from the backbone. In eitherconformation, the pendant segments will normally depend from a modifiednucleotide or other organic moiety having the appropriate functionalgroups for attachment of oligonucleotides. Furthermore, in eitherconformation, a large number of amplification sequences are availablefor binding, either directly or indirectly, to detection probes. Ingeneral, these structures are made as is known in the art, usingmodified multifunctional nucleotides, as is described in U.S. Pat. Nos.5,635,352 and 5,124,246, among others.

In a preferred embodiment, dendrimer amplifier probes are used, as aregenerally described in U.S. Pat. No. 5,175,270, hereby expresslyincorporated by reference. Dendrimeric amplifier probes haveamplification sequences that are attached via hybridization, and thushave portions of double-stranded nucleic acid as a component of theirstructure. The outer surface of the dendrimer amplifier probe has amultiplicity of amplification sequences.

In a preferred embodiment, linear amplifier probes are used, that haveindividual amplification sequences linked end-to-end either directly orwith short intervening sequences to form a polymer. As with the otheramplifier configurations, there may be additional sequences or moietiesbetween the amplification sequences. In addition, as outlined herein,linear amplification probes may form hairpin stem-loop structures, as isdepicted in FIG. 12.

In one embodiment, the linear amplifier probe has a single amplificationsequence. This may be useful when cycles of hybridization/disassociationoccurs, forming a pool of amplifier probe that was hybridized to thetarget and then removed to allow more probes to bind, or when largenumbers of ETMs are used for each label probe. However, in a preferredembodiment, linear amplifier probes comprise a multiplicity ofamplification sequences.

In addition, the amplifier probe may be totally linear, totallybranched, totally dendrimeric, or any combination thereof.

The amplification sequences of the amplifier probe are used, eitherdirectly or indirectly, to bind to a label probe to allow detection. Ina preferred embodiment, the amplification sequences of the amplifierprobe are substantially complementary to a first portion of a labelprobe. Alternatively, amplifier extender probes are used, that have afirst portion that binds to the amplification sequence and a secondportion that binds to the first portion of the label probe.

In addition, the compositions of the invention may include“preamplifier” molecules, which serves a bridging moiety between thelabel extender molecules and the amplifier probes. In this way, moreamplifier and thus more ETMs are ultimately bound to the detectionprobes. Preamplifier molecules may be either linear or branched, andtypically contain in the range of about 30-3000 nucleotides.

The reactions outlined below may be accomplished in a variety of ways,as will be appreciated by those in the art. Components of the reactionmay be added simultaneously, or sequentially, in any order, withpreferred embodiments outlined below. In addition, the reaction mayinclude a variety of other reagents may be included in the assays. Theseinclude reagents like salts, buffers, neutral proteins, e.g. albumin,detergents, etc which may be used to facilitate optimal hybridizationand detection, and/or reduce non-specific or background interactions.Also reagents that otherwise improve the efficiency of the assay, suchas protease inhibitors, nuclease inhibitors, anti-microbial agents,etc., may be used, depending on the sample preparation methods andpurity of the target. Generally, the methods are as follows. In apreferred embodiment, the target is initially driven down to thevicinity of the detection probe using any one of the methods outlinedabove. In general, two methods may be employed; the assay complexes asdescribed below are formed first (i.e. all the soluble components areadded together, either simultaneously or sequentially, including captureextender probes, label probes, amplification probes, label extenderprobes, etc.), including any hybridization accelerators, and then thecomplex is added to the surface for binding to a detection electrode.Alternatively, the target may be added, hybridization accelerationoccurs to allow the target to bind the capture binding ligand and thenadditional components are added to form the assay complex. The latter isdescribed in detail below, but either procedure may be followed.Similarly, some components may be added, electrophoresed, and othercomponents added; for example, the target analyte may be combined withany capture extender probes and then transported, etc. In addition, asoutlined herein, electrophoretic steps may be used to effect “washing”steps, wherein excess reagents (non-bound analytes, excess probes, etc.)can be driven from the surface.

In a preferred embodiment, non-specific interactions can be decreasedusing several electrophoretic methods. In a preferred embodiment, labelprobes that are not specifically directly or indirectly bound to atarget sequence can be removed from the surface by a pulse of anopposite electric field, i.e. the electric field is reversed for someperiod of time. The strength of the reverse electric field is chosensuch that specifically bound label probes are not removed (or any of theother required components of the attachment and assay complexes).

In a preferred embodiment, for example when electrophoresis is used, thelabel probes or label binding ligands comprising the ETMs carry a chargeopposite to the target analyte. This can be done either with nucleicacid label probes or charged solution binding ligands, although thediscussion focuses on nucleic acid embodiments. This can be useful intwo different systems. In a preferred embodiment, the target analytecarries a large excess of charge, i.e. a negative charge in the case ofnucleic acid. The binding of one or more positively charged label probesdoes not significantly change the net negative charge on the targetcomplex; that is, the target will still be attracted to the cathode.However, un-bound label probes, or label probes not specifically boundto the target, are repulsed, thus resulting in a decrease ofnon-specific binding. For example, PNA backbones can be modified tocarry a net positive charge, and there are other nucleic acid analogs asknown in the art that are positively charged.

In a preferred embodiment, the label probe is has a high amount ofopposite charge, such that upon binding to the target analyte, the netcharge of the target analyte changes. Thus, for example, for nucleicacids, the label probes carry a sufficient positive charge to render thelabel probe-target analyte complex positively charged. This results inthe specific target analyte being drawn to the anode, but all othernegatively charged elements, i.e. other nucleic acids, will be repulsed.This is particularly useful when there is an excess of other targetspresent; for example, when the target analyte is a minor species of alarge excess of other nucleic acids, for example.

In a preferred embodiment, the target analyte is initiallyelectrophoretically transported to the detection electrode, and thenimmobilized or attached to the detection electrode. In one embodiment,this is done by forming an attachment complex (frequently referred toherein as a hybridization complex when nucleic acid components are used)between a capture probe and a portion of the target analyte. A preferredembodiment utilizes capture extender binding ligands (also calledcapture extender probes herein); in this embodiment, an attachmentcomplex is formed between a portion of the target sequence and a firstportion of a capture extender probe, and an additional attachmentcomplex between a second portion of the capture extender probe and aportion of the capture probe. Additional preferred embodiments utilizeadditional capture probes, thus forming an attachment complex between aportion of the target sequence and a first portion of a second captureextender probe, and an attachment complex between a second portion ofthe second capture extender probe and a second portion of the captureprobe.

Alternatively, the attachment of the target sequence to the electrode isdone simultaneously with the other reactions.

The method proceeds with the introduction of amplifier probes, ifutilized. In a preferred embodiment, the amplifier probe comprises afirst probe sequence that is substantially complementary to a portion ofthe target sequence, and at least one amplification sequence.

In one embodiment, the first probe sequence of the amplifier probe ishybridized to the target sequence, and any unhybridized amplifier probeis removed. This will generally be done as is known in the art, anddepends on the type of assay. When the target sequence is immobilized ona surface such as an electrode, the removal of excess reagents generallyis done via one or more washing steps, as will be appreciated by thosein the art. In this embodiment, the target may be immobilized on anysolid support. When the target sequence is not immobilized on a surface,the removal of excess reagents such as the probes of the invention maybe done by adding beads (i.e. solid support particles) that containcomplementary sequences to the probes, such that the excess probes bindto the beads. The beads can then be removed, for example bycentrifugation, filtration, the application of magnetic or electrostaticfields, etc.

The reaction mixture is then subjected to conditions (temperature, highsalt, changes in pH, etc.) under which the amplifier probe disassociatesfrom the target sequence, and the amplifier probe is collected. Theamplifier probe may then be added to an electrode comprising captureprobes for the amplifier probes, label probes added, and detection isachieved.

In a preferred embodiment, a larger pool of probe is generated by addingmore amplifier probe to the target sequence and thehybridization/disassociation reactions are repeated, to generate alarger pool of amplifier probe. This pool of amplifier probe is thenadded to an electrode comprising amplifier capture probes, label probesadded, and detection proceeds.

In this embodiment, it is preferred that the target sequence beimmobilized on a solid support, including an electrode, using themethods described herein; although as will be appreciated by those inthe art, alternate solid support attachment technologies may be used,such as attachment to glass, polymers, etc. It is possible to do thereaction on one solid support and then add the pooled amplifier probe toan electrode for detection.

In a preferred embodiment, the amplifier probe comprises a multiplicityof amplification sequences.

In one embodiment, the first probe sequence of the amplifier probe ishybridized to the target sequence, and any unhybridized amplifier probeis removed. Again, preferred embodiments utilize immobilized targetsequences, wherein the target sequences are immobilized by hybridizationwith capture probes that are attached to the electrode, or hybridizationto capture extender probes that in turn hybridize with immobilizedcapture probes as is described herein. Generally, in these embodiments,the capture probes and the detection probes are immobilized on theelectrode, generally at the same “address”.

In a preferred embodiment, the first probe sequence of the amplifierprobe is hybridized to a first portion of at least one label extenderprobe, and a second portion of the label extender probe is hybridized toa portion of the target sequence. Other preferred embodiments utilizemore than one label extender probe, as is generally shown in FIG. 5G.

In a preferred embodiment, the amplification sequences of the amplifierprobe are used directly for detection, by hybridizing at least one labelprobe sequence.

The invention thus provides assay complexes that minimally comprise atarget sequence and a label probe. “Assay complex” herein is meant thecollection of attachment or hybridization complexes comprising analytes,including binding ligands and targets, that allows detection. Thecomposition of the assay complex depends on the use of the differentprobe component outlined herein. Thus, in FIG. 6A, the assay complexcomprises the capture probe and the target sequence. The assay complexesmay also include capture extender probes, label extender probes, andamplifier probes, as outlined herein, depending on the configurationused.

The assays are generally run under stringency conditions which allowsformation of the label probe attachment complex only in the presence oftarget. Stringency can be controlled by altering a step parameter thatis a thermodynamic variable, including, but not limited to, temperature,formamide concentration, salt concentration, chaotropic saltconcentration pH, organic solvent concentration, etc. Stringency mayalso include the use of an electrophoretic step to drive non-specific(i.e. low stringency) materials away from the detection electrode.

These parameters may also be used to control non-specific binding, as isgenerally outlined in U.S. Pat. No. 5,681,697. Thus it may be desirableto perform certain steps at higher stringency conditions; for example,when an initial hybridization step is done between the target sequenceand the label extender and capture extender probes. Running this step atconditions which favor specific binding can allow the reduction ofnon-specific binding.

In a preferred nucleic acid embodiment, when all of the componentsoutlined herein are used, a preferred method is as follows.Single-stranded target sequence is incubated under hybridizationconditions with the capture extender probes and the label extenderprobes. A preferred embodiment does this reaction in the presence of theelectrode with immobilized capture probes, although this may also bedone in two steps, with the initial incubation and the subsequentaddition to the electrode. Excess reagents are washed off, and amplifierprobes are then added. If preamplifier probes are used, they may beadded either prior to the amplifier probes or simultaneously with theamplifier probes. Excess reagents are washed off, and label probes arethen added. Excess reagents are washed off, and detection proceeds asoutlined below.

In one embodiment, a number of capture probes (or capture probes andcapture extender probes) that are each substantially complementary to adifferent portion of the target sequence are used.

Again, as outlined herein, when amplifier probes are used, the system isgenerally configured such that upon label probe binding, the recruitmentlinkers comprising the ETMs are placed in proximity either to themonolayer surface containing conductive oligomers (mechanism-2) or inproximity to detection probes. Thus for example, for mechanism-2systems, when the ETMs are attached via “dendrimer” type structures asoutlined herein, the length of the linkers from the nucleic acid pointof attachment to the ETMs may vary, particularly with the length of thecapture probe when capture extender probes are used. That is, longercapture probes, with capture extenders, can result in the targetsequences being “held” further away from the surface than for shortercapture probes. Adding extra linking sequences between the probe nucleicacid and the ETMs can result in the ETMs being spatially closer to thesurface, giving better results. Similarly, for mechanism-1 systems, thelength of the recruitment linker, the length of the detection probe, andtheir distance, may be optimized.

In addition, if desirable, nucleic acids utilized in the invention mayalso be ligated together prior to detection, if applicable, by usingstandard molecular biology techniques such as the use of a ligase.Similarly, if desirable for stability, cross-linking agents may be addedto hold the structures stable.

As will be appreciated by those in the art, while described for nucleicacids, the systems outlined herein can be used for other target analytesas well.

The compositions of the invention are generally synthesized as outlinedbelow and in U.S. Ser. Nos. 08/743,798, 08/873,978, 08/911,085,08/911,085, and PCT US97/20014, all of which are expressly incorporatedby reference, generally utilizing techniques well known in the art. Aswill be appreciated by those in the art, many of the techniques outlinedbelow are directed to nucleic acids containing a ribose-phosphatebackbone. However, as outlined above, many alternate nucleic acidanalogs may be utilized, some of which may not contain either ribose orphosphate in the backbone. In these embodiments, for attachment atpositions other than the base, attachment is done as will be appreciatedby those in the art, depending on the backbone. Thus, for example,attachment can be made at the carbon atoms of the PNA backbone, as isdescribed below, or at either terminus of the PNA.

The compositions may be made in several ways. A preferred method firstsynthesizes a conductive oligomer attached to a nucleoside, withaddition of additional nucleosides to form the capture probe followed byattachment to the electrode. Alternatively, the whole capture probe maybe made and then the completed conductive oligomer added, followed byattachment to the electrode. Alternatively, a monolayer of conductiveoligomer (some of which have functional groups for attachment of captureprobes) is attached to the electrode first, followed by attachment ofthe capture probe. The latter two methods may be preferred whenconductive oligomers are used which are not stable in the solvents andunder the conditions used in traditional nucleic acid synthesis.

In a preferred embodiment, the compositions of the invention are made byfirst forming the conductive oligomer covalently attached to thenucleoside, followed by the addition of additional nucleosides to form acapture probe nucleic acid, with the last step comprising the additionof the conductive oligomer to the electrode.

The attachment of the conductive oligomer to the nucleoside may be donein several ways. In a preferred embodiment, all or part of theconductive oligomer is synthesized first (generally with a functionalgroup on the end for attachment to the electrode), which is thenattached to the nucleoside. Additional nucleosides are then added asrequired, with the last step generally being attachment to theelectrode. Alternatively, oligomer units are added one at a time to thenucleoside, with addition of additional nucleosides and attachment tothe electrode. A number of representative syntheses are shown in theFigures of WO 98/20162; PCT/US98/12430; PCT/US98/12082; PCT/US99/01705;PCT/US99/01703; and U.S. Ser. Nos. 09/135,183; 60/105,875; and09/295,691, all of which are incorporated by reference.

The conductive oligomer is then attached to a nucleoside that maycontain one (or more) of the oligomer units, attached as depictedherein.

In a preferred embodiment, attachment is to a ribose of theribose-phosphate backbone, including amide and amine linkages. In apreferred embodiment, there is at least a methylene group or other shortaliphatic alkyl groups (as a Z group) between the nitrogen attached tothe ribose and the aromatic ring of the conductive oligomer.

Alternatively, attachment is via a phosphate of the ribose-phosphatebackbone, as generally outlined in PCT US97/20014.

In a preferred embodiment, attachment is via the base. In a preferredembodiment, protecting groups may be added to the base prior to additionof the conductive oligomers, as is generally known in the art. Inaddition, the palladium cross-coupling reactions may be altered toprevent dimerization problems; i.e. two conductive oligomers dimerizing,rather than coupling to the base.

Alternatively, attachment to the base may be done by making thenucleoside with one unit of the oligomer, followed by the addition ofothers.

Once the modified nucleosides are prepared, protected and activated,prior to attachment to the electrode, they may be incorporated into agrowing oligonucleotide by standard synthetic techniques (Gait,Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford, UK1984; Eckstein) in several ways.

In one embodiment, one or more modified nucleosides are converted to thetriphosphate form and incorporated into a growing oligonucleotide chainby using standard molecular biology techniques such as with the use ofthe enzyme DNA polymerase 1, T4 DNA polymerase, T7 DNA polymerase, TaqDNA polymerase, reverse transcriptase, and RNA polymerases. For theincorporation of a 3′ modified nucleoside to a nucleic acid, terminaldeoxynucleotidyltransferase may be used. (Ratliff, Terminaldeoxynucleotidyltransferase. In The Enzymes, Vol 14A. P. D. Boyer ed. pp105-118. Academic Press, San Diego, Calif. 1981). Thus, the presentinvention provides deoxyribonucleoside triphosphates comprising acovalently attached ETM. Preferred embodiments utilize ETM attachment tothe base or the backbone, such as the ribose (preferably in the 2′position), as is generally depicted below in Structures 42 and 43:

Thus, in some embodiments, it may be possible to generate the nucleicacids comprising ETMs in situ. For example, a target sequence canhybridize to a capture probe (for example on the surface) in such a waythat the terminus of the target sequence is exposed, i.e. unhybridized.The addition of enzyme and triphosphate nucleotides labelled with ETMsallows the in situ creation of the label. Similarly, using labelednucleotides recognized by polymerases can allow simultaneous PCR anddetection; that is, the target sequences are generated in situ.

In a preferred embodiment, the modified nucleoside is converted to thephosphoramidite or H-phosphonate form, which are then used insolid-phase or solution syntheses of oligonucleotides. In this way themodified nucleoside, either for attachment at the ribose (i.e. amino- orthiol-modified nucleosides) or the base, is incorporated into theoligonucleotide at either an internal position or the 5′ terminus. Thisis generally done in one of two ways. First, the 5′ position of theribose is protected with 4′,4-dimethoxytrityl (DMT) followed by reactionwith either 2-cyanoethoxy-bis-diisopropylaminophosphine in the presenceof diisopropylammonium tetrazolide, or by reaction withchlorodiisopropylamino 2′-cyanoethyoxyphosphine, to give thephosphoramidite as is known in the art; although other techniques may beused as will be appreciated by those in the art. See Gait, supra;Caruthers, Science 230:281 (1985), both of which are expresslyincorporated herein by reference.

For attachment of a group to the 3′ terminus, a preferred methodutilizes the attachment of the modified nucleoside (or the nucleosidereplacement) to controlled pore glass (CPG) or other oligomericsupports. In this embodiment, the modified nucleoside is protected atthe 5′ end with DMT, and then reacted with succinic anhydride withactivation. The resulting succinyl compound is attached to CPG or otheroligomeric supports as is known in the art. Further phosphoramiditenucleosides are added, either modified or not, to the 5′ end afterdeprotection. Thus, the present invention provides conductive oligomersor insulators covalently attached to nucleosides attached to solidoligomeric supports such as CPG, and phosphoramidite derivatives of thenucleosides of the invention.

The invention further provides methods of making label probes withrecruitment linkers comprising ETMs. These synthetic reactions willdepend on the character of the recruitment linker and the method ofattachment of the ETM, as will be appreciated by those in the art. Fornucleic acid recruitment linkers, the label probes are generally made asoutlined herein with the incorporation of ETMs at one or more positions.When a transition metal complex is used as the ETM, synthesis may occurin several ways. In a preferred embodiment, the ligand(s) are added to anucleoside, followed by the transition metal ion, and then thenucleoside with the transition metal complex attached is added to anoligonucleotide, i.e. by addition to the nucleic acid synthesizer.Alternatively, the ligand(s) may be attached, followed by incorportationinto a growing oligonucleotide chain, followed by the addition of themetal ion.

In a preferred embodiment, ETMs are attached to a ribose of theribose-phosphate backbone. This is generally done as is outlined hereinfor conductive oligomers, as described herein, and in PCT publication WO95/15971, using amino-modified or oxo-modified nucleosides, at eitherthe 2′ or 3′ position of the ribose. The amino group may then be usedeither as a ligand, for example as a transition metal ligand forattachment of the metal ion, or as a chemically functional group thatcan be used for attachment of other ligands or organic ETMs, for examplevia amide linkages, as will be appreciated by those in the art. Forexample, the examples describe the synthesis of nucleosides with avariety of ETMs attached via the ribose.

In a preferred embodiment, ETMs are attached to a phosphate of theribose-phosphate backbone. As outlined herein, this may be done usingphosphodiester analogs such as phosphoramidite bonds, see generally PCTpublication WO 95/15971, or can be done in a similar manner to thatdescribed in PCT US97/20014, where the conductive oligomer is replacedby a transition metal ligand or complex or an organic ETM.

Attachment to alternate backbones, for example peptide nucleic acids oralternate phosphate linkages will be done as will be appreciated bythose in the art.

In a preferred embodiment, ETMs are attached to a base of thenucleoside. This may be done in a variety of ways. In one embodiment,amino groups of the base, either naturally occurring or added as isdescribed herein (see the fiigures, for example), are used either asligands for transition metal complexes or as a chemically functionalgroup that can be used to add other ligands, for example via an amidelinkage, or organic ETMs. This is done as will be appreciated by thosein the art. Alternatively, nucleosides containing halogen atoms attachedto the heterocyclic ring are commercially available. Acetylene linkedligands may be added using the halogenated bases, as is generally known;see for example, Tzalis et al., Tetrahedron Lett. 36(34):60176020(1995); Tzalis et al., Tetrahedron Lett. 36(2):3489-3490 (1995); andTzalis et al., Chem. Communications (in press) 1996, all of which arehereby expressly incorporated by reference. See also the figures and theexamples, which describes the synthesis of metallocenes (in this case,ferrocene) attached via acetylene linkages to the bases.

In one embodiment, the nucleosides are made with transition metalligands, incorporated into a nucleic acid, and then the transition metalion and any remaining necessary ligands are added as is known in theart. In an alternative embodiment, the transition metal ion andadditional ligands are added prior to incorporation into the nucleicacid.

Once the nucleic acids of the invention are made, with a covalentlyattached attachment linker (i.e. either an insulator or a conductiveoligomer), the attachment linker is attached to the electrode. Themethod will vary depending on the type of electrode used. As isdescribed herein, the attachment linkers are generally made with aterminal “A” linker to facilitate attachment to the electrode. For thepurposes of this application, a sulfur-gold attachment is considered acovalent attachment.

In a preferred embodiment, conductive oligomers, insulators, andattachment linkers are covalently attached via sulfur linkages to theelectrode. However, surprisingly, traditional protecting groups for useof attaching molecules to gold electrodes are generally not ideal foruse in both synthesis of the compositions described herein and inclusionin oligonucleotide synthetic reactions. Accordingly, the presentinvention provides novel methods for the attachment of conductiveoligomers to gold electrodes, utilizing unusual protecting groups,including ethylpyridine, and trimethylsilylethyl as is depicted in theFigures. However, as will be appreciated by those in the art, when theconductive oligomers do not contain nucleic acids, traditionalprotecting groups such as acetyl groups and others may be used. SeeGreene et al., supra.

This may be done in several ways. In a preferred embodiment, the subunitof the conductive oligomer which contains the sulfur atom for attachmentto the electrode is protected with an ethyl-pyridine ortrimethylsilylethyl group. For the former, this is generally done bycontacting the subunit containing the sulfur atom (preferably in theform of a sulfhydryl) with a vinyl pyridine group or vinyltrimethylsilylethyl group under conditions whereby an ethylpyridinegroup or trimethylsilylethyl group is added to the sulfur atom.

This subunit also generally contains a functional moiety for attachmentof additional subunits, and thus additional subunits are attached toform the conductive oligomer. The conductive oligomer is then attachedto a nucleoside, and additional nucleosides attached. The protectinggroup is then removed and the sulfur-gold covalent attachment is made.Alternatively, all or part of the conductive oligomer is made, and theneither a subunit containing a protected sulfur atom is added, or asulfur atom is added and then protected. The conductive oligomer is thenattached to a nucleoside, and additional nucleosides attached.Alternatively, the conductive oligomer attached to a nucleic acid ismade, and then either a subunit containing a protected sulfur atom isadded, or a sulfur atom is added and then protected. Alternatively, theethyl pyridine protecting group may be used as above, but removed afterone or more steps and replaced with a standard protecting group like adisulfide. Thus, the ethyl pyridine or trimethylsilylethyl group mayserve as the protecting group for some of the synthetic reactions, andthen removed and replaced with a traditional protecting group.

By “subunit” of a conductive polymer herein is meant at least the moietyof the conductive oligomer to which the sulfur atom is attached,although additional atoms may be present, including either functionalgroups which allow the addition of additional components of theconductive oligomer, or additional components of the conductiveoligomer. Thus, for example, when Structure 1 oligomers are used, asubunit comprises at least the first Y group.

A preferred method comprises 1) adding an ethyl pyridine ortrimethylsilylethyl protecting group to a sulfur atom attached to afirst subunit of a conductive oligomer, generally done by adding a vinylpyridine or trimethylsilylethyl group to a sulfhydryl; 2) addingadditional subunits to form the conductive oligomer; 3) adding at leasta first nucleoside to the conductive oligomer; 4) adding additionalnucleosides to the first nucleoside to form a nucleic acid; 5) attachingthe conductive oligomer to the gold electrode. This may also be done inthe absence of nucleosides, as is described in the Examples.

The above method may also be used to attach insulator molecules to agold electrode.

In a preferred embodiment, a monolayer comprising conductive oligomers(and optionally insulators) is added to the electrode. Generally, thechemistry of addition is similar to or the same as the addition ofconductive oligomers to the electrode, i.e. using a sulfur atom forattachment to a gold electrode, etc. Compositions comprising monolayersin addition to the conductive oligomers covalently attached to nucleicacids may be made in at least one of five ways: (1) addition of themonolayer, followed by subsequent addition of the attachmentlinker-nucleic acid complex; (2) addition of the attachmentlinker-nucleic acid complex followed by addition of the monolayer; (3)simultaneous addition of the monolayer and attachment linker-nucleicacid complex; (4) formation of a monolayer (using any of 1, 2 or 3)which includes attachment linkers which terminate in a functional moietysuitable for attachment of a completed nucleic acid; or (5) formation ofa monolayer which includes attachment linkers which terminate in afunctional moiety suitable for nucleic acid synthesis, i.e. the nucleicacid is synthesized on the surface of the monolayer as is known in theart. Such suitable functional moieties include, but are not limited to,nucleosides, amino groups, carboxyl groups, protected sulfur moieties,or hydroxyl groups for phosphoramidite additions. The examples describethe formation of a monolayer on a gold electrode using the preferredmethod (1).

In a preferred embodiment, the nucleic acid is a peptide nucleic acid oranalog. In this embodiment, the invention provides peptide nucleic acidswith at least one covalently attached ETM or attachment linker. In apreferred embodiment, these moieties are covalently attached to anmonomeric subunit of the PNA. By “monomeric subunit of PNA” herein ismeant the —NH—CH₂CH₂—N(COCH₂-Base)—CH₂—CO— monomer, or derivatives(herein included within the definition of “nucleoside”) of PNA. Forexample, the number of carbon atoms in the PNA backbone may be altered;see generally Nielsen et al., Chem. Soc. Rev. 1997 page 73, whichdiscloses a number of PNA derivatives, herein expressly incorporated byreference. Similarly, the amide bond linking the base to the backbonemay be altered; phosphoramide and sulfuramide bonds may be used.Alternatively, the moieties are attached to an internal monomericsubunit. By “internal” herein is meant that the monomeric subunit is noteither the N-terminal monomeric subunit or the C-terminal monomericsubunit. In this embodiment, the moieties can be attached either to abase or to the backbone of the monomeric subunit. Attachment to the baseis done as outlined herein or known in the literature. In general, themoieties are added to a base which is then incorporated into a PNA asoutlined herein. The base may be either protected, as required forincorporation into the PNA synthetic reaction, or derivatized, to allowincorporation, either prior to the addition of the chemical substituentor afterwards. Protection and derivatization of the bases is shown inPCT US97/20014. The bases can then be incorporated into monomericsubunits.

In a preferred embodiment, the moieties are covalently attached to thebackbone of the PNA monomer. The attachment is generally to one of theunsubstituted carbon atoms of the monomeric subunit, preferably thea-carbon of the backbone, although attachment at either of the carbon 1or 2 positions, or the a-carbon of the amide bond linking the base tothe backbone may be done. In the case of PNA analogs, other carbons oratoms may be substituted as well. In a preferred embodiment, moietiesare added at the a-carbon atoms, either to a terminal monomeric subunitor an internal one.

In this embodiment, a modified monomeric subunit is synthesized with anETM or an attachment linker, or a functional group for its attachment,and then the base is added and the modified monomer can be incorporatedinto a growing PNA chain.

Once generated, the monomeric subunits with covalently attached moietiesare incorporated into a PNA using the techniques outlined in Will etal., Tetrahedron 51(44):12069-12082 (1995), and Vanderlaan et al., Tett.Let. 38:2249-2252 (1997), both of which are hereby expresslyincorporated in their entirety. These procedures allow the addition ofchemical substituents to peptide nucleic acids without destroying thechemical substituents.

As will be appreciated by those in the art, electrodes may be made thathave any combination of nucleic acids, conductive oligomers andinsulators.

The compositions of the invention may additionally contain one or morelabels at any position. By “label” herein is meant an element (e.g. anisotope) or chemical compound that is attached to enable the detectionof the compound. Preferred labels are radioactive isotopic labels, andcolored or fluorescent dyes. The labels may be incorporated into thecompound at any position. In addition, the compositions of the inventionmay also contain other moieties such as cross-linking agents tofacilitate cross-linking of the target-probe complex. See for example,Lukhtanov et al., Nucl. Acids. Res. 24(4):683 (1996) and Tabone et al.,Biochem. 33:375 (1994), both of which are expressly incorporated byreference.

Once made, the compositions find use in a number of applications, asdescribed herein. In particular, the compositions of the invention finduse in binding assays for the detection of target analytes, inparticular nucleic acid target sequences. As will be appreciated bythose in the art, electrodes can be made that have a single species ofbinding ligand, or multiple binding ligand species, i.e. in an arrayformat.

In addition, as outlined herein, the use of a solid support such as anelectrode enables the use of these assays in an array form. For example,the use of oligonucleotide arrays are well known in the art. Inaddition, techniques are known for “addressing” locations within anelectrode and for the surface modification of electrodes. Thus, in apreferred embodiment, arrays of different binding ligands, includingnucleic acids, are laid down on the electrode, each of which arecovalently attached to the electrode via an attachment linker. In thisembodiment, the number of different binding ligands may vary widely,from one to thousands, with from about 4 to about 100,000 beingpreferred, and from about 10 to about 10,000 being particularlypreferred.

Once the assay complexes of the invention are made, that minimallycomprise a target analyte and a label probe, detection proceeds withelectronic initiation. Without being limited by the mechanism or theory,detection is based on the transfer of electrons from the ETM to theelectrode, including via the “π-way”.

Detection of electron transfer, i.e. the presence of the ETMs, isgenerally initiated electronically, with voltage being preferred. Apotential is applied to the assay complex. Precise control andvariations in the applied potential can be via a potentiostat and eithera three electrode system (one reference, one sample (or working) and onecounter electrode) or a two electrode system (one sample and one counterelectrode). This allows matching of applied potential to peak potentialof the system which depends in part on the choice of ETMs and in part onthe conductive oligomer used, the composition and integrity of themonolayer, and what type of reference electrode is used. As describedherein, ferrocene is a preferred ETM.

In a preferred embodiment, a co-reductant or co-oxidant (collectively,co-redoxant) is used, as an additional electron source or sink. Seegenerally Sato et al., Bull. Chem. Soc. Jpn 66:1032 (1993); Uosaki etal., Electrochimica Acta 36:1799 (1991); and Alleman et al., J. Phys.Chem 100:17050 (1996); all of which are incorporated by reference.

In a preferred embodiment, an input electron source in solution is usedin the initiation of electron transfer, preferably when initiation anddetection are being done using DC current or at AC frequencies wherediffusion is not limiting. In general, as will be appreciated by thosein the art, preferred embodiments utilize monolayers that contain aminimum of “holes”, such that short-circuiting of the system is avoided.This may be done in several general ways. In a preferred embodiment, aninput electron source is used that has a lower or similar redoxpotential than the ETM of the label probe. Thus, at voltages above theredox potential of the input electron source, both the ETM and the inputelectron source are oxidized and can thus donate electrons; the ETMdonates an electron to the electrode and the input source donates to theETM. For example, ferrocene, as a ETM attached to the compositions ofthe invention as described in the examples, has a redox potential ofroughly 200 mV in aqueous solution (which can change significantlydepending on what the ferrocene is bound to, the manner of the linkageand the presence of any substitution groups). Ferrocyanide, an electronsource, has a redox potential of roughly 200 mV as well (in aqueoussolution). Accordingly, at or above voltages of roughly 200 mV,ferrocene is converted to ferricenium, which then transfers an electronto the electrode. Now the ferricyanide can be oxidized to transfer anelectron to the ETM. In this way, the electron source (or co-reductant)serves to amplify the signal generated in the system, as the electronsource molecules rapidly and repeatedly donate electrons to the ETMattached to the nucleic acid. The rate of electron donation oracceptance will be limited by the rate of diffusion of the co-reductant,the electron transfer between the co-reductant and the ETM, which inturn is affected by the concentration and size, etc.

Alternatively, input electron sources that have lower redox potentialsthan the ETM are used. At voltages less than the redox potential of theETM, but higher than the redox potential of the electron source, theinput source such as ferrocyanide is unable to be oxided and thus isunable to donate an electron to the ETM; i.e. no electron transferoccurs. Once ferrocene is oxidized, then there is a pathway for electrontransfer.

In an alternate preferred embodiment, an input electron source is usedthat has a higher redox potential than the ETM of the label probe. Forexample, luminol, an electron source, has a redox potential of roughly720 mV. At voltages higher than the redox potential of the ETM, butlower than the redox potential of the electron source, i.e. 200-720 mV,the ferrocene is oxided, and transfers a single electron to theelectrode via the conductive oligomer. However, the ETM is unable toaccept any electrons from the luminol electron source, since thevoltages are less than the redox potential of the luminol. However, ator above the redox potential of luminol, the luminol then transfers anelectron to the ETM, allowing rapid and repeated electron transfer. Inthis way, the electron source (or co-reductant) serves to amplify thesignal generated in the system, as the electron source molecules rapidlyand repeatedly donate electrons to the ETM of the label probe.

Luminol has the added benefit of becoming a chemiluminiscent speciesupon oxidation (see Jirka et al., Analytica Chimica Acta 284:345(1993)), thus allowing photo-detection of electron transfer from the ETMto the electrode. Thus, as long as the luminol is unable to contact theelectrode directly, i.e. in the presence of the SAM such that there isno efficient electron transfer pathway to the electrode, luminol canonly be oxidized by transferring an electron to the ETM on the labelprobe. When the ETM is not present, i.e. when the target sequence is nothybridized to the composition of the invention, luminol is notsignificantly oxidized, resulting in a low photon emission and thus alow (if any) signal from the luminol. In the presence of the target, amuch larger signal is generated. Thus, the measure of luminol oxidationby photon emission is an indirect measurement of the ability of the ETMto donate electrons to the electrode. Furthermore, since photondetection is generally more sensitive than electronic detection, thesensitivity of the system may be increased. Initial results suggest thatluminescence may depend on hydrogen peroxide concentration, pH, andluminol concentration, the latter of which appears to be non-linear.

Suitable electron source molecules are well known in the art, andinclude, but are not limited to, ferricyanide, and luminol.

Alternatively, output electron acceptors or sinks could be used, i.e.the above reactions could be run in reverse, with the ETM such as ametallocene receiving an electron from the electrode, converting it tothe metallicenium, with the output electron acceptor then accepting theelectron rapidly and repeatedly. In this embodiment, cobalticenium isthe preferred ETM.

The presence of the ETMs at the surface of the monolayer can be detectedin a variety of ways. A variety of detection methods may be used,including, but not limited to, optical detection (as a result ofspectral changes upon changes in redox states), which includesfluorescence, phosphorescence, luminiscence, chemiluminescence,electrochemiluminescence, and refractive index; and electronicdetection, including, but not limited to, amperommetry, voltammetry,capacitance and impedence. These methods include time or frequencydependent methods based on AC or DC currents, pulsed methods, lock-intechniques, filtering (high pass, low pass, band pass), andtime-resolved techniques including time-resolved fluorescence.

In one embodiment, the efficient transfer of electrons from the ETM tothe electrode results in stereotyped changes in the redox state of theETM. With many ETMs including the complexes of ruthenium containingbipyridine, pyridine and imidazole rings, these changes in redox stateare associated with changes in spectral properties. Significantdifferences in absorbance are observed between reduced and oxidizedstates for these molecules. See for example Fabbrizzi et al., Chem. Soc.Rev. 1995 pp197-202). These differences can be monitored using aspectrophotometer or simple photomultiplier tube device.

In this embodiment, possible electron donors and acceptors include allthe derivatives listed above for photoactivation or initiation.Preferred electron donors and acceptors have characteristically largespectral changes upon oxidation and reduction resulting in highlysensitive monitoring of electron transfer. Such examples includeRu(NH₃)₄py and Ru(bpy)₂im as preferred examples. It should be understoodthat only the donor or acceptor that is being monitored by absorbanceneed have ideal Espectral characteristics.

In a preferred embodiment, the electron transfer is detectedfluorometrically. Numerous transition metal complexes, including thoseof ruthenium, have distinct fluorescence properties. Therefore, thechange in redox state of the electron donors and electron acceptorsattached to the nucleic acid can be monitored very sensitively usingfluorescence, for example with Ru(4,7-biphenyl₂-phenanthroline)₃ ²⁺. Theproduction of this compound can be easily measured using standardfluorescence assay techniques. For example, laser induced fluorescencecan be recorded in a standard single cell fluorimeter, a flow through“on-line” fluorimeter (such as those attached to a chromatographysystem) or a multi-sample “plate-reader” similar to those marketed for96-well immuno assays.

Alternatively, fluorescence can be measured using fiber optic sensorswith nucleic acid probes in solution or attached to the fiber optic.Fluorescence is monitored using a photomultiplier tube or other lightdetection instrument attached to the fiber optic. The advantage of thissystem is the extremely small volumes of sample that can be assayed.

In addition, scanning fluorescence detectors such as the Fluorlmagersold by Molecular Dynamics are ideally suited to monitoring thefluorescence of modified nucleic acid molecules arrayed on solidsurfaces. The advantage of this system is the large number of electrontransfer probes that can be scanned at once using chips covered withthousands of distinct nucleic acid probes.

Many transition metal complexes display fluorescence with large Stokesshifts. Suitable examples include bis- and trisphenanthroline complexesand bis- and trisbipyridyl complexes of transition metals such asruthenium (see Juris, A., Balzani, V., et. al. Coord. Chem. Rev., V. 84,p. 85-277, 1988). Preferred examples display efficient fluorescence(reasonably high quantum yields) as well as low reorganization energies.These include Ru(4,7-biphenyl₂-phenanthroline)₃ ²⁺,Ru(4,4′-diphenyl-2,2′-bipyridine)₃ ²⁺ and platinum complexes (seeCummings et al., J. Am. Chem. Soc. 118:1949-1960 (1996), incorporated byreference). Alternatively, a reduction in fluorescence associated withhybridization can be measured using these systems.

In a further embodiment, electrochemiluminescence is used as the basisof the electron transfer detection. With some ETMs such as Ru²⁺ (bpy)₃,direct luminescence accompanies excited state decay. Changes in thisproperty are associated with nucleic acid hybridization and can bemonitored with a simple photomultiplier tube arrangement (see Blackburn,G. F. Clin. Chem. 37: 1534-1539 (1991); and Juris et al., supra.

In a preferred embodiment, electronic detection is used, includingamperommetry, voltammetry, capacitance, and impedence. Suitabletechniques include, but are not limited to, electrogravimetry;coulometry (including controlled potential coulometry and constantcurrent coulometry); voltametry (cyclic voltametry, pulse voltametry(normal pulse voltametry, square wave voltametry, differential pulsevoltametry, Osteryoung square wave voltametry, and coulostatic pulsetechniques); stripping analysis (aniodic stripping analysis, cathiodicstripping analysis, square wave stripping voltammetry); conductancemeasurements (electrolytic conductance, direct analysis); time-dependentelectrochemical analyses (chronoamperometry, chronopotentiometry, cyclicchronopotentiometry and amperometry, AC polography, chronogalvametry,and chronocoulometry); AC impedance measurement; capacitancemeasurement; AC voltametry; and photoelectrochemistry.

In a preferred embodiment, monitoring electron transfer is viaamperometric detection. This method of detection involves applying apotential (as compared to a separate reference electrode) between thenucleic acid-conjugated electrode and a reference (counter) electrode inthe sample containing target genes of interest. Electron transfer ofdiffering efficiencies is induced in samples in the presence or absenceof target nucleic acid; that is, the presence or absence of the targetnucleic acid, and thus the label probe, can result in differentcurrents.

The device for measuring electron transfer amperometrically involvessensitive current detection and includes a means of controlling thevoltage potential, usually a potentiostat. This voltage is optimizedwith reference to the potential of the electron donating complex on thelabel probe. Possible electron donating complexes include thosepreviously mentioned with complexes of iron, osmium, platinum, cobalt,rhenium and ruthenium being preferred and complexes of iron being mostpreferred.

In a preferred embodiment, alternative electron detection modes areutilized. For example, potentiometric (or voltammetric) measurementsinvolve non-faradaic (no net current flow) processes and are utilizedtraditionally in pH and other ion detectors. Similar sensors are used tomonitor electron transfer between the ETM and the electrode. Inaddition, other properties of insulators (such as resistance) and ofconductors (such as conductivity, impedance and capicitance) could beused to monitor electron transfer between ETM and the electrode.Finally, any system that generates a current (such as electron transfer)also generates a small magnetic field, which may be monitored in someembodiments.

It should be understood that one benefit of the fast rates of electrontransfer observed in the compositions of the invention is that timeresolution can greatly enhance the signal-to-noise results of monitorsbased on absorbance, fluorescence and electronic current. The fast ratesof electron transfer of the present invention result both in highsignals and stereotyped delays between electron transfer initiation andcompletion. By amplifying signals of particular delays, such as throughthe use of pulsed initiation of electron transfer and “lock-in”amplifiers of detection, and Fourier transforms.

In a preferred embodiment, electron transfer is initiated usingalternating current (AC) methods. Without being bound by theory, itappears that ETMs, bound to an electrode, generally respond similarly toan AC voltage across a circuit containing resistors and capacitors.Basically, any methods which enable the determination of the nature ofthese complexes, which act as a resistor and capacitor, can be used asthe basis of detection. Surprisingly, traditional electrochemicaltheory, such as exemplified in Laviron et al., J. Electroanal. Chem.97:135 (1979) and Laviron et al., J. Electroanal. Chem. 105:35 (1979),both of which are incorporated by reference, do not accurately model thesystems described herein, except for very small E_(AC) (less than 10 mV)and relatively large numbers of molecules. That is, the AC current (I)is not accurately described by Laviron's equation. This may be due inpart to the fact that this theory assumes an unlimited source and sinkof electrons, which is not true in the present systems.

The AC voltametry theory that models these systems well is outlined inO'Connor et al., J. Electroanal. Chem. 466(2):197-202 (1999), herebyexpressly incorporated by reference. The equation that predicts thesesystems is shown below as Equation 1: $\begin{matrix}{i_{avg} = {2n\quad f\quad F\quad {N_{total} \cdot \frac{\sinh \quad\left\lbrack {\frac{n\quad F}{R\quad T} \cdot E_{AC}} \right\rbrack}{{\cosh \quad\left\lbrack {\frac{n\quad F}{R\quad T} \cdot E_{AC}} \right\rbrack} + {\cosh \quad\left\lbrack {\frac{n\quad F}{R\quad T}\quad \left( {E_{DC} - E_{O}} \right)} \right\rbrack}}}}} & {{Equation}\quad 1}\end{matrix}$

In Equation 1, n is the number of electrons oxidized or reduced perredox molecule, f is the applied frequency, F is Faraday's constant,N_(total) is the total number of redox molecules, E_(O) is the formalpotential of the redox molecule, R is the gas constant, T is thetemperature in degrees Kelvin, and E_(DC) is the electrode potential.The model fits the experimental data very well. In some cases thecurrent is smaller than predicted, however this has been shown to becaused by ferrocene degradation which may be remedied in a number ofways.

In addition, the faradaic current can also be expressed as a function oftime, as shown in Equation 2: $\begin{matrix}{{I_{f}(t)} = {\frac{q_{e}N_{total}n\quad F}{2R\quad T\quad \left( {{\cosh \quad\left\lbrack {\frac{n\quad F}{R\quad T}\quad \left( {{V(t)} - E_{O}} \right)} \right\rbrack} + 1} \right)} \cdot \frac{{V(t)}}{t}}} & {{Equation}\quad 2}\end{matrix}$

I_(F) is the Faradaic current and q_(e) is the elementary charge.

However, Equation 1 does not incorporate the effect of electron transferrate nor of instrument factors. Electron transfer rate is important whenthe rate is close to or lower than the applied frequency. Thus, the truei_(AC) should be a function of all three, as depicted in Equation 3.

i _(AC) =f(Nernst factors)f(k _(ET))f(instrument factors)  Equation 3

These equations can be used to model and predict the expected ACcurrents in systems which use input signals comprising both AC and DCcomponents. As outlined above, traditional theory surprisingly does notmodel these systems at all, except for very low voltages.

In general, non-specifically bound label probes/ETMs show differences inimpedance (i.e. higher impedances) than when the label probes containingthe ETMs are specifically bound in the correct orientation. In apreferred embodiment, the non-specifically bound material is washedaway, resulting in an effective impedance of infinity. Thus, ACdetection gives several advantages as is generally discussed below,including an increase in sensitivity, and the ability to “filter out”background noise. In particular, changes in impedance (including, forexample, bulk impedance) as between non-specific binding ofETM-containing probes and target-specific assay complex formation may bemonitored.

Accordingly, when using AC initiation and detection methods, thefrequency response of the system changes as a result of the presence ofthe ETM. By “frequency response” herein is meant a modification ofsignals as a result of electron transfer between the electrode and theETM. This modification is different depending on signal frequency. Afrequency response includes AC currents at one or more frequencies,phase shifts, DC offset voltages, faradaic impedance, etc.

Once the assay complex including the target sequence and label probe ismade, a first input electrical signal is then applied to the system,preferably via at least the sample electrode (containing the complexesof the invention) and the counter electrode, to initiate electrontransfer between the electrode and the ETM. Three electrode systems mayalso be used, with the voltage applied to the reference and workingelectrodes. The first input signal comprises at least an AC component.The AC component may be of variable amplitude and frequency. Generally,for use in the present methods, the AC amplitude ranges from about 1 mVto about 1.1 V, with from about 10 mV to about 800 mV being preferred,and from about 10 mV to about 500 mV being especially preferred. The ACfrequency ranges from about 0.01 Hz to about 100 MHz, with from about 10Hz to about 10 MHz being preferred, and from about 100 Hz to about 20MHz being especially preferred.

The use of combinations of AC and DC signals gives a variety ofadvantages, including surprising sensitivity and signal maximization.

In a preferred embodiment, the first input signal comprises a DCcomponent and an AC component. That is, a DC offset voltage between thesample and counter electrodes is swept through the electrochemicalpotential of the ETM (for example, when ferrocene is used, the sweep isgenerally from 0 to 500 mV) (or alternatively, the working electrode isgrounded and the reference electrode is swept from 0 to −500 mV). Thesweep is used to identify the DC voltage at which the maximum responseof the system is seen. This is generally at or about the electrochemicalpotential of the ETM. Once this voltage is determined, either a sweep orone or more uniform DC offset voltages may be used. DC offset voltagesof from about −1 V to about +1.1 V are preferred, with from about −500mV to about +800 mV being especially preferred, and from about −300 mVto about 500 mV being particularly preferred. In a preferred embodiment,the DC offset voltage is not zero. On top of the DC offset voltage, anAC signal component of variable amplitude and frequency is applied. Ifthe ETM is present, and can respond to the AC perturbation, an ACcurrent will be produced due to electron transfer between the electrodeand the ETM.

For defined systems, it may be sufficient to apply a single input signalto differentiate between the presence and absence of the ETM (i.e. thepresence of the target sequence) nucleic acid. Alternatively, aplurality of input signals are applied. As outlined herein, this maytake a variety of forms, including using multiple frequencies, multipleDC offset voltages, or multiple AC amplitudes, or combinations of any orall of these.

Thus, in a preferred embodiment, multiple DC offset voltages are used,although as outlined above, DC voltage sweeps are preferred. This may bedone at a single frequency, or at two or more frequencies .

In a preferred embodiment, the AC amplitude is varied. Without beingbound by theory, it appears that increasing the amplitude increases thedriving force. Thus, higher amplitudes, which result in higheroverpotentials give faster rates of electron transfer. Thus, generally,the same system gives an improved response (i.e. higher output signals)at any single frequency through the use of higher overpotentials at thatfrequency. Thus, the amplitude may be increased at high frequencies toincrease the rate of electron transfer through the system, resulting ingreater sensitivity. In addition, this may be used, for example, toinduce responses in slower systems such as those that do not possessoptimal spacing configurations.

In a preferred embodiment, measurements of the system are taken at atleast two separate amplitudes or overpotentials, with measurements at aplurality of amplitudes being preferred. As noted above, changes inresponse as a result of changes in amplitude may form the basis ofidentification, calibration and quantification of the system. Inaddition, one or more AC frequencies can be used as well.

In a preferred embodiment, the AC frequency is varied. At differentfrequencies, different molecules respond in different ways. As will beappreciated by those in the art, increasing the frequency generallyincreases the output current. However, when the frequency is greaterthan the rate at which electrons may travel between the electrode andthe ETM, higher frequencies result in a loss or decrease of outputsignal. At some point, the frequency will be greater than the rate ofelectron transfer between the ETM and the electrode, and then the outputsignal will also drop.

In one embodiment, detection utilizes a single measurement of outputsignal at a single frequency. That is, the frequency response of thesystem in the absence of target sequence, and thus the absence of labelprobe containing ETMs, can be previously determined to be very low at aparticular high frequency. Using this information, any response at aparticular frequency, will show the presence of the assay complex. Thatis, any response at a particular frequency is characteristic of theassay complex. Thus, it may only be necessary to use a single input highfrequency, and any changes in frequency response is an indication thatthe ETM is present, and thus that the target sequence is present.

In addition, the use of AC techniques allows the significant reductionof background signals at any single frequency due to entities other thanthe ETMs, i.e. “locking out” or “filtering” unwanted signals. That is,the frequency response of a charge carrier or redox active molecule insolution will be limited by its diffusion coefficient and chargetransfer coefficient. Accordingly, at high frequencies, a charge carriermay not diffuse rapidly enough to transfer its charge to the electrode,and/or the charge transfer kinetics may not be fast enough. This isparticularly significant in embodiments that do not have goodmonolayers, i.e. have partial or insufficient monolayers, i.e. where thesolvent is accessible to the electrode. As outlined above, in DCtechniques, the presence of “holes” where the electrode is accessible tothe solvent can result in solvent charge carriers “short circuiting” thesystem, i.e. the reach the electrode and generate background signal.However, using the present AC techniques, one or more frequencies can bechosen that prevent a frequency response of one or more charge carriersin solution, whether or not a monolayer is present. This is particularlysignificant since many biological fluids such as blood containsignificant amounts of redox active molecules which can interfere withamperometric detection methods.

In a preferred embodiment, measurements of the system are taken at atleast two separate frequencies, with measurements at a plurality offrequencies being preferred. A plurality of frequencies includes a scan.For example, measuring the output signal, e.g., the AC current, at a lowinput frequency such as 1-20 Hz, and comparing the response to theoutput signal at high frequency such as 10-100 kHz will show a frequencyresponse difference between the presence and absence of the ETM. In apreferred embodiment, the frequency response is determined at at leasttwo, preferably at least about five, and more preferably at least aboutten frequencies.

After transmitting the input signal to initiate electron transfer, anoutput signal is received or detected. The presence and magnitude of theoutput signal will depend on a number of factors, including theoverpotential/amplitude of the input signal; the frequency of the inputAC signal; the composition of the intervening medium; the DC offset; theenvironment of the system; the nature of the ETM; the solvent; and thetype and concentration of salt. At a given input signal, the presenceand magnitude of the output signal will depend in general on thepresence or absence of the ETM, the placement and distance of the ETMfrom the surface of the monolayer and the character of the input signal.In some embodiments, it may be possible to distinguish betweennon-specific binding of label probes and the formation of targetspecific assay complexes containing label probes, on the basis ofimpedance. In a preferred embodiment, the output signal comprises an ACcurrent. As outlined above, the magnitude of the output current willdepend on a number of parameters. By varying these parameters, thesystem may be optimized in a number of ways.

In general, AC currents generated in the present invention range fromabout 1 femptoamp to about 1 milliamp, with currents from about 50femptoamps to about 100 microamps being preferred, and from about 1picoamp to about 1 microamp being especially preferred.

In a preferred embodiment, the output signal is phase shifted in the ACcomponent relative to the input signal. Without being bound by theory,it appears that the systems of the present invention may be sufficientlyuniform to allow phase-shifting based detection. That is, the complexbiomolecules of the invention through which electron transfer occursreact to the AC input in a homogeneous manner, similar to standardelectronic components, such that a phase shift can be determined. Thismay serve as the basis of detection between the presence and absence ofthe ETM, and/or differences between the presence of target-specificassay complexes comprising label probes and non-specific binding of thelabel probes to the system components.

The output signal is characteristic of the presence of the ETM; that is,the output signal is characteristic of the presence of thetarget-specific assay complex comprising label probes and ETMs. In apreferred embodiment, the basis of the detection is a difference in thefaradaic impedance of the system as a result of the formation of theassay complex. Faradaic impedance is the impedance of the system betweenthe electrode and the ETM. Faradaic impedance is quite different fromthe bulk or dielectric impedance, which is the impedance of the bulksolution between the electrodes. Many factors may change the faradaicimpedance which may not effect the bulk impedance, and vice versa. Thus,the assay complexes comprising the nucleic acids in this system have acertain faradaic impedance, that will depend on the distance between theETM and the electrode, their electronic properties, and the compositionof the intervening medium, among other things. Of importance in themethods of the invention is that the faradaic impedance between the ETMand the electrode is signficantly different depending on whether thelabel probes containing the ETMs are specifically or non-specificallybound to the electrode.

Accordingly, the present invention further provides electronic devicesor apparatus for the detection of analytes using the compositions of theinvention. The apparatus includes a test chamber for receiving a samplesolution which has at least a first measuring or sample electrode, and asecond measuring or counter electrode. Three electrode systems are alsouseful. The first and second measuring electrodes are in contact with atest sample receiving region, such that in the presence of a liquid testsample, the two electrophoresis electrodes may be in electrical contact.

In a preferred embodiment, the apparatus also includes detectionelectrodes comprising a single stranded nucleic acid capture probecovalently attached via an attachment linker, and a monolayer comprisingconductive oligomers, such as are described herein.

The apparatus further comprises an AC voltage source electricallyconnected to the test chamber; that is, to the measuring electrodes.Preferably, the AC voltage source is capable of delivering DC offsetvoltage as well.

In a preferred embodiment, the apparatus further comprises a processorcapable of comparing the input signal and the output signal. Theprocessor is coupled to the electrodes and configured to receive anoutput signal, and thus detect the presence of the target nucleic acid.

Thus, the compositions of the present invention may be used in a varietyof research, clinical, quality control, or field testing settings.

In a preferred embodiment, the probes are used in genetic diagnosis. Forexample, probes can be made using the techniques disclosed herein todetect target sequences such as the gene for nonpolyposis colon cancer,the BRCA1 breast cancer gene, P53, which is a gene associated with avariety of cancers, the Apo E4 gene that indicates a greater risk ofAlzheimer's disease, allowing for easy presymptomatic screening ofpatients, mutations in the cystic fibrosis gene, or any of the otherswell known in the art.

In an additional embodiment, viral and bacterial detection is done usingthe complexes of the invention. In this embodiment, probes are designedto detect target sequences from a variety of bacteria and viruses. Forexample, current blood-screening techniques rely on the detection ofanti-HIV antibodies. The methods disclosed herein allow for directscreening of clinical samples to detect HIV nucleic acid sequences,particularly highly conserved HIV sequences. In addition, this allowsdirect monitoring of circulating virus within a patient as an improvedmethod of assessing the efficacy of anti-viral therapies. Similarly,viruses associated with leukemia, HTLV-I and HTLV-II, may be detected inthis way. Bacterial infections such as tuberculosis, clymidia and othersexually transmitted diseases, may also be detected.

In a preferred embodiment, the nucleic acids of the invention find useas probes for toxic bacteria in the screening of water and food samples.For example, samples may be treated to lyse the bacteria to release itsnucleic acid, and then probes designed to recognize bacterial strains,including, but not limited to, such pathogenic strains as, Salmonella,Campylobacter, Vibrio cholerae, Leishmania, enterotoxic strains of E.coli, and Legionnaire's disease bacteria. Similarly, bioremediationstrategies may be evaluated using the compositions of the invention.

In a further embodiment, the probes are used for forensic “DNAfingerprinting” to match crime-scene DNA against samples taken fromvictims and suspects.

In an additional embodiment, the probes in an array are used forsequencing by hybridization.

Thus, the present invention provides for extremely specific andsensitive probes, which may, in some embodiments, detect targetsequences without removal of unhybridized probe. This will be useful inthe generation of automated gene probe assays.

Alternatively, the compositions of the invention are useful to detectsuccessful gene amplification in PCR, thus allowing successful PCRreactions to be an indication of the presence or absence of a targetsequence. PCR may be used in this manner in several ways. For example,in one embodiment, the PCR reaction is done as is known in the art, andthen added to a composition of the invention comprising the targetnucleic acid with a ETM, covalently attached to an electrode via aconductive oligomer with subsequent detection of the target sequence.Alternatively, PCR is done using nucleotides labelled with a ETM, eitherin the presence of, or with subsequent addition to, an electrode with aconductive oligomer and a target nucleic acid. Binding of the PCRproduct containing ETMs to the electrode composition will allowdetection via electron transfer. Finally, the nucleic acid attached tothe electrode via a conductive polymer may be one PCR primer, withaddition of a second primer labelled with an ETM. Elongation results indouble stranded nucleic acid with a ETM and electrode covalentlyattached. In this way, the present invention is used for PCR detectionof target sequences.

In a preferred embodiment, the arrays are used for mRNA detection. Apreferred embodiment utilizes either capture probes or capture extenderprobes that hybridize close to the 3′ polyadenylation tail of the mRNAs.This allows the use of one species of target binding probe fordetection, i.e. the probe contains a poly-T portion that will bind tothe poly-A tail of the mRNA target. Generally, the probe will contain asecond portion, preferably non-poly-T, that will bind to the detectionprobe (or other probe). This allows one target-binding probe to be made,and thus decreases the amount of different probe synthesis that is done.

In a preferred embodiment, the use of restriction enzymes and ligationmethods allows the creation of “universal” arrays. In this embodiment,monolayers comprising capture probes that comprise restrictionendonuclease ends, as is generally depicted in FIG. 6. By utilizingcomplementary portions of nucleic acid, while leaving “sticky ends”, anarray comprising any number of restriction endonuclease sites is made.Treating a target sample with one or more of these restrictionendonucleases allows the targets to bind to the array. This can be donewithout knowing the sequence of the target. The target sequences can beligated, as desired, using standard methods such as ligases, and thetarget sequence detected, using either standard labels or the methods ofthe invention.

The present invention provides methods which can result in sensitivedetection of nucleic acids. In a preferred embodiment, less than about10×10⁶ molecules are detected, with less than about 10×10⁵ beingpreferred, less than 10×10⁴ being particularly preferred, less thanabout 10×10³ being especially preferred, and less than about 10×10²being most preferred. As will be appreciated by those in the art, thisassumes a 1:1 correlation between target sequences and reportermolecules; if more than one reporter molecule (i.e. electron transfermoeity) is used for each target sequence, the sensitivity will go up.

While the limits of detection are currently being evaluated, based onthe published electron transfer rate through DNA, which is roughly 1×10⁶electrons/sec/duplex for an 8 base pair separation (see Meade et al.,Angw. Chem. Eng. Ed., 34:352 (1995)) and high driving forces, ACfrequencies of about 100 kHz should be possible. As the preliminaryresults show, electron transfer through these systems is quiteefficient, resulting in nearly 100×10³ electrons/sec, resulting inpotential femptoamp sensitivity for very few molecules.

All references cited herein are incorporated by reference in theirentireity.

EXAMPLES Example 1 General Methods of Making Substrates and Monolayers

SAM formation on Substrates-General Procedure

The self-assembled monolayers were formed on a clean gold surface. Thegold surface can be prepared by a variety of different methods: meltedor polished gold wire, sputtered or evaporated gold on glass or mica orsilicon wafers or some other substrate, electroplated or electrolessgold on circuit board material or glass or silicon or some othersubstrate. Both the vacuum deposited gold samples (evaporated andsputtered) and the solution deposited gold samples (electroless andelectroplated) often require the use of an adhesion layer between thesubstrate and the gold in order to insure good mechanical stability.Chromium, Titanium, Titanium/Tungsten or Tantalum is frequently employedwith sputtered and evaporated gold. Electroplated nickel is usuallyemployed with electroplated and electroless gold, however other adhesionmaterials can be used.

The gold substrate is cleaned prior to monolayer formation. A variety ofdifferent procedures have been employed. Cleaning with a chemicalsolution is the most prevalent. Piranha solution (hydrogenperoxide/sulfuric acid) or aqua regia cleaning (Hydrochloric acid/Nitricacid) is most prevalent, however electrochemical methods, flametreatment and plasma methods have also been employed.

Following cleaning, the gold substrate is incubated in a depositionsolution. The deposition solution consists of a mixture of variousthiols in a solvent. A mixture of alkane thiols in an organic solventlike ethanol is the most prevalent procedure, however numerousvariations have been developed. Alternative procedures involve gas phasedeposition of the alkane thiol, microcontact printing, deposition usingneat thiol, deposition from aqueous solvent and two step procedures havebeen developed. The concentration of the alkane thiol in the depositionsolution ranges from molar to submicromolar range with 0.5-2.0millimolar being the most prevalent. The gold substrate isincubated/placed in contact with the deposition solution for less than asecond to days depending on the procedure. The most common time is lhrto overnight incubation. The incubation is usually performed at roomtemperature, however temperatures up to 50° C. are common.

Mixed monolayers that contain DNA are usually prepared using a two stepprocedure. The thiolated DNA is deposited during the first depositionstep and the mixed monolayer formation is completed during the secondstep in which a second thiol solution minus DNA is added. The secondstep frequently involves mild heating to promote monolayerreorganization.

General Procedure for SAM formation-Deposited from Organic Solution

A clean gold surface was placed into a clean vial. A DNA depositionsolution in organic solvent was prepared in which the total thiolconcentration was between 400 uM and 1.0 mM. The deposition solutioncontained thiol modified DNA and thiol diluent molecules. The ratio ofDNA to diluent was usually between 10:1 and 1:10 with 1:1 beingpreferred. The preferred solvents are tetrahydrofuran (THF),acetonitrile, dimethylforamide (DMF) or mixtures thereof. Sufficient DNAdeposition solution is added to the vial so as to completely cover theelectrode surface. The gold substrate is allowed to incubate at ambienttemperature or slightly above ambient temperature for 5-30 minutes.After the initial incubation, the deposition solution is removed and asolution of diluent molecule only (100 uM-1.0 mM) in organic solvent isadded. The gold substrate is allowed to incubate at room temperature orabove room temperature until a complete monolayer is formed (10minutes-24 hours). The gold sample is removed from the solution, rinsedin clean solvent and used.

General Procedure for SAM formation-deposited from Aqueous Solution

A clean gold surface is placed into a clean vial. A DNA depositionsolution in water is prepared in which the total thiol concentration isbetween 1 uM and 200 uM. The aqueous solution frequently has saltpresent (approximately 1M), however pure water can be used. Thedeposition solution contains thiol modified DNA and often a thioldiluent molecule. The ratio of DNA to diluent is usually between 10:1and 1:10 with 1:1 being preferred. The DNA deposition solution is addedto the vial in such a volume so as to completely cover the electrodesurface. The gold substrate is allowed to incubate at ambienttemperature or slightly above ambient temperature for 1-30 minutes with5 minutes usually being sufficient. After the initial incubation, thedeposition solution is removed and a solution of diluent molecule only(10 uM-1.0 mM) in either water or organic solvent is added. The goldsubstrate is allowed to incubate at room temperature or above roomtemperature until a complete monolayer is formed (10 minutes-24 hours).The gold sample is removed from the solution, rinsed in clean solventand used.

Monolayers on Au Ball Electrodes

Creating Au Ball Electrodes: Use a razor blade to cut 10 cm lengths ofgold wire (127 μm diameter, 99.99% pure; e.g. from Aldrich). Use a 16gauge needle to pass the wire through a #4 natural rubber septum (of thesize to fit over a ½ mL PCR eppendorf tube). (This serves to support thewire and seal the tubes during deposition. See below.) Use aclean-burning flame (methane or propane) to melt one centimeter of thewire and form a sphere attached to the wire terminus. Adjust the wirelength such that when sealed in a PCR tube the gold ball would bepositioned near the bottom, able to be submerged in 20 μL of liquid. Onthe day of use, dip the electrodes in aqua regia (4:3:1 H₂O:HCl:HNO₃)for 20 seconds and then rinse thoroughly with water.

Derivatization: For 5 minutes, heat 20 μL aliquots of depositionsolutions (2:2:1 DNA/H6/M44 at 833 μM total in DMF) in PCR tubes on aPCR block at 50° C. Then put each electrode into a tube of depositionsolution (submerging just the gold ball—as little of the wire “stem” aspossible) and remove to room temperature. Incubate for fifteen minutesbefore transferring the electrodes into PCR tubes with 200 μL of 400 μMM44 in DMF (submerging much of the wire stem as well). Let sit in M44 atroom temperature for 5 minutes, then put on the PCR block and runHCLONG. Take electrodes out of the M44 solution, dip in 6×SSC, and placein PCR tubes with 20 μL of hybridization solution. Dip electrodes in6×SSC prior to ACV measurement.

HCLONG: 65° C. 2′, −0.3° C./s to 40° C., 40° C. 2′, +0.3° C./s to 55°C., 55° C. 2′, −0.3° C./s to 30° C., 30° C. 2′, +0.3° C./s to 35° C.,35° C. 2′, −0.3° C/.s to 22° C.

Manufacture of Circuit Boards

An 18″×24″×0.047″ panel of FR-4 (General Electric) with a half-ouncecopper foil on both sides was drilled according to specifications(Gerber files). The FR-4 panel is plated with electroless copper (500microinches) to make the specified drill-holes conductive and then panelis plated with an additional 500 microinches of electroplated copper.Following copper plating, the panel is etched according tospecifications via cupric chloride etching (acid etching). The etchedpanel is then plated with 400 microinches of electroplated nickel withbrightner followed by 50 microinches of soft gold (99.99% purity). Thegold panel is coated with liquid photoimagable solder mask (Probimer 52,Ciba-Geigy Co.) on both sides of the panel. The imaging is doneaccording to specifications. 14 sensor electrodes that are 250 micron indiameter and 2 larger electrodes (500 microns in diameter) are createdwith insulated leads leading to gold plated contacts at the edge of theboard. The solder masked panel is then scored according tospecifications to create individual wafers that are 1″×1″. Asilver/silver chloride paste is applied to one of the two largerelectrodes (ERCON R414). The panel is then plasma cleaned with anArgon/Oxygen Plasma mixture. Following cleaning, the panel is stored ina foil-lined bag until use.

Monolayer Deposition on Circuit Boards

The circuit boards are removed from the foil-lined bags and immersed ina 10% sulfuric acid solution for 30 seconds. Following the sulfuric acidtreatment, the boards are immersed in two Milli-Q water baths for 1minute each. The boards are then dried under a stream of nitrogen. Theboards are placed on a X-Y table in a humidity chamber and a 30nanoliter drop of DNA deposition solution is placed on each of the 14electrodes. The DNA deposition solution consists of 33 uM thiolated DNA,33 uM 2-unit phenylacetylene wire (H6), and 16 uM M44 in 6×SSC (900 mMsodium chloride, 90 mM sodium Citrate, pH 7) w/1% Triethylamine. Thedrop is incubated at room temperature for 5 minutes and then the drop isremoved by rinsing in a Milli-Q water bath. The boards are immersed in a45° C. bath of M44 in acetontrile. After 30 minutes, the boards areremoved and immersed in an acetonitrile bath for 30 seconds followed bya milli-Q water bath for 30 seconds. The boards are dried under a streamof nitrogen.

Example 2 Detection of Target Sequences

Monolayer Deposition on Circuit Boards

As above, the circuit boards were removed from the foil-lined bags andimmersed in a 10% sulfuric acid solution for 30 seconds. Following thesulfuric acid treatment, the boards were immersed in two Milli-Q waterbaths for 1 minute each. The boards were then dried under a stream ofnitrogen. The boards were placed on a X-Y table in a humidity chamberand a 30 nanoliter drop of DNA deposition solution was placed on each ofthe 14 electrodes. The DNA deposition solution consisted of 33 uMthiolated DNA, 33 uM 2-unit phenylacetylene wire (H6), and 16 uMundec-1-en-11yltri(ethylene glycol)(HS-CH₂)₁₁—(OCH₂CH₂)₃—OH) in 6×SSC(900 mM sodium chloride, 90 mM sodium Citrate, pH 7) w/1% Triethylamine.3 electrodes were spotted with a solution containing DNA 1(5′-ACCATGGACACAGAT(CH₂)₁₆SH-3′). 4 electrodes were spotted with asolution containing DNA 2 (5′TCATTGATGGTCTCTTTTAACA((CH₂)₁₆SH-3′). 4electrodes were spotted with DNA 3(5′CACAGTGGGGGGACATCMGCAGCCATGCAM(CH₂)₁₆SH-3′). 3 electrodes werespotted with DNA 4 (5′-TGTGCAGTTGACGTGGAT(CH₂)₁₆SH-3′). The depositionsolution was allowed to incubate at room temperature for 5 minutes andthen the drop was removed by rinsing in a Milli-Q water bath. The boardswere immersed in a 45° C. bath of M44 in acetonitrile. After 30 minutes,the boards were removed and immersed in an acetonitrile bath for 30seconds followed by a milli-Q water bath for 30 seconds. The boards weredried under a stream of nitrogen and stored in foiled-lined bags flushedwith nitrogen until use.

Hybridization and Measurement

The modified boards were removed from the foil-lined bags and fittedwith an injection molded sample chamber (cartridge). The chamber wasadhered to the board using double-sided sticky tape and had a totalvolume of 250 microliters. A hybridization solution was prepared. Thesolution contains 10 nM DNA target(5′-TGTGCAGTTGACGTGGATTGTTAAAAGAGACCATCAATGAGGMGCTGCAGMTGGGATAGAGTCATCCAGT-3′ (D-998), 30 nM signaling probe (D-1055) and 10nm 5′-TCTACAG(N6)C(N6)ATCTGTGTCCATGGT-3′ (N6 is shown in FIG. 1D ofPCTUS99/01705; it comprises a ferrocene connected by a 4 carbon chain tothe 2′ oxygen of the ribose of a nucleoside). The signalling probe is asfollows:

5′-(C23)₄-N87-N87-N87-N87-ATC CAC GTC AAC TGC ACA-3′ (D-1055)            |    |   |   |            C23  C23 C23 C23           C23  C23 C23 C23            C23  C23 C23 C23           C23  C23 C23 C23

N87 is a branch point comprising a ring structure. C23 is shown in FIG.1 F of PCTUS99/01705. In a solution containing 25% Qiagen lysis bufferAL, 455 mM NaClO₄, 195 mM NaCl, 1.0 mM mercaptohexanol and 10% fetalcalf serum. 250 microliters of hybrid solution was injected into thecartridge and allowed to hybridize for 12 hours. After 12 hours, thehybridized chip was plugged into a homemade transconductance amplifierwith switching circuitry. The transconductance amplifier was equippedwith summing circuitry that combines a DC ramp from the computer DAQcard and an AC sine wave from the lock-in amplifier (SR830 StanfordInstruments). Each electrode was scanned sequentially and the data wassaved and manipulated using a homemade program designed using Labview(National Instruments). The chip was scanned at between −100 mV and 500mV (pseudo Ag/Ag/Cl reference electrode) DC with a 25 mV (50 mV peak topeak), 1000 Hz superimposed sine wave. The output current was fed intothe lock-in amplifier and the 1000 Hz signal was recorded (ACVtechnique). The data for each set of pads was compiled and averaged.

lp Relative intensity lp DNA 1 (Positive 2 Fc) 34 nA 0.11 DNA 2(Positive Sandwich Assay) 218 nA 0.7 DNA 3 (Negative) 0.3 nA 0.001 DNA 4(Positive Sandwich Assay) 317 nA 1

The results are shown in FIG. 14.

We claim:
 1. A method of detecting a target analyte in a samplecomprising: a) concentrating said target analyte in a detection chambercomprising a detection electrode comprising a self-assembled monolayerand a covalently attached capture ligand; b) binding said target analyteto said capture ligand to form an assay complex, wherein said assaycomplex further comprises at least one electron transfer moiety (ETM);and c) detecting the presence of said ETM using said detectionelectrode.
 2. A method according to claim 1 wherein said concentratingis done by placing said sample in an electric field between at least afirst electrode and at least a second electrode sufficient to causeelectrophoretic transport of said sample to said detection electrode. 3.A method according to claim 2 wherein said detection electrode is aporous electrode positioned between said first and said secondelectrodes.
 4. A method according to claim 2 wherein said detectionelectrode is the same as said first electrode.
 5. A method according toclaim 4 wherein at least said first electrode comprises a permeationlayer.
 6. A method according to claim 4 wherein an electroactive chargecarrier is used for said electrophoretic transport.
 7. A methodaccording to claim 2 wherein said detection electrode is separate fromsaid first or second electrodes.
 8. A method according to claim 1wherein said assay complex comprises a label probe comprising said ETM,and said label probe is positively charged.
 9. A method according toclaim 1 wherein said concentrating comprises including at least onevolume exclusion agent in said detection chamber.
 10. A method accordingto claim 1 wherein said concentrating comprises precipitating saidtarget analyte.
 11. A method according to claim 1 wherein saidconcentrating comprises including at least two reagents that form twoseparable solution phases, such that said target analyte concentrates inone of said phases.
 12. A method according to claim 1 wherein saidconcentrating comprises binding said target analyte to a shuttleparticle.
 13. A method of detecting a target analyte in a samplecomprising: a) flowing said sample past a detection electrode comprisinga self-assembled monolayer and a covalently attached capture ligandunder conditions that result in the formation of an assay complexcomprising said target analyte and said capture ligand, wherein saidassay complex further comprises at least one electron transfer moiety(ETM); and c) detecting the presence of said ETM using said detectionelectrode.
 14. A method according to claim 13 wherein the configurationof said detection electrode results in mixing of said sample.
 15. Amethod according to claim 14 wherein said detection electrode comprisesweirs.
 16. A method according to claim 13 wherein said detectionelectrode is porous and is positioned such that said sample flowsthrough said electrode.
 17. A method of detecting a target nucleic acidsequence in a sample comprising: a) indirectly or directly hybridizingsaid target sequence to a capture probe covalently attached to adetection electrode comprising a self-assembled monolayer to form anassay complex, wherein said assay complex is formed in the presence of ahybridization accelerator, wherein said assay complex further comprisesat least one electron transfer moiety (ETM); and b) detecting thepresence of said ETM using said detection electrode.
 18. A methodaccording to claim 17 wherein said hybridization accelerator is anucleic acid binding protein.
 19. A method according to claim 17 whereinsaid hybridization accelerator is a polyvalent ion.
 20. A method ofdetecting a target analyte in a sample comprising: a) adding said sampleto a detection electrode comprising a covalently attached capture ligandunder conditions that result in the formation of an assay complexcomprising said target analyte and said capture ligand, wherein saidconditions include the presence of mixing particles, wherein said assaycomplex further comprises at least one electron transfer moiety (ETM);b) detecting the presence of said ETM using said detection electrode.21. A method of detecting a target analyte in a sample comprising: a)concentrating said target analyte in a detection chamber comprising adetection electrode comprising a covalently attached capture ligand,wherein said concentrating is done by placing said sample in an electricfield between at least a first electrode and at least a second electrodesufficient to cause electrophoretic transport of said sample to saiddetection electrode and said detection electrode is a porous electrodepositioned between said first and said second electrodes; b) bindingsaid target analyte to said capture ligand to form an assay complex,wherein said assay complex further comprises at least one electrontransfer moiety (ETM); and c) detecting the presence of said ETM usingsaid detection electrode.
 22. A method of detecting a target analyte ina sample comprising: a) concentrating said target analyte in a detectionchamber comprising a detection electrode comprising a covalentlyattached capture ligand; b) binding said target analyte to said captureligand to form an assay complex, wherein said assay complex furthercomprises at least one electron transfer moiety (ETM) and a label probecomprising said ETM, and said label probe is positively charged; and c)detecting the presence of said ETM using said detection electrode.
 23. Amethod of detecting a target analyte in a sample comprising: a)concentrating said target analyte in a detection chamber comprising adetection electrode comprising a covalently attached capture ligand,wherein said concentrating comprises including at least one volumeexclusion agent in said detection chamber; b) binding said targetanalyte to said capture ligand to form an assay complex, wherein saidassay complex further comprises at least one electron transfer moiety(ETM); and c) detecting the presence of said ETM using said detectionelectrode.
 24. A method of detecting a target analyte in a samplecomprising: a) concentrating said target analyte in a detection chambercomprising a detection electrode comprising a covalently attachedcapture ligand, wherein said concentrating comprises precipitating saidtarget analyte; b) binding said target analyte to said capture ligand toform an assay complex, wherein said assay complex further comprises atleast one electron transfer moiety (ETM); and c) detecting the presenceof said ETM using said detection electrode.
 25. A method of detecting atarget analyte in a sample comprising: a) concentrating said targetanalyte in a detection chamber comprising a detection electrodecomprising a covalently attached capture ligand, wherein saidconcentrating comprises including at least two reagents that form twoseparable solution phases, such that said target analyte concentrates inone of said phases; b) binding said target analyte to said captureligand to form an assay complex, wherein said assay complex furthercomprises at least one electron transfer moiety (ETM); and c) detectingthe presence of said ETM using said detection electrode.
 26. A method ofdetecting a target analyte in a sample comprising: a) concentrating saidtarget analyte in a detection chamber comprising a detection electrodecomprising a covalently attached capture ligand, wherein saidconcentrating comprises binding said target analyte t o a shuttleparticle; b) binding said target analyte to said capture ligand to forman assay complex, wherein said assay complex further comprises at leastone electron transfer moiety (ETM); and c) detecting the presence ofsaid ETM using said detection electrode.
 27. A method of detecting atarget analyte in a sample comprising: a) flowing said sample past adetection electrode comprising a covalently attached capture ligandunder conditions that result in the formation of an assay complexcomprising said target analyte and said capture ligand, wherein saidassay complex further comprises at least one electron transfer moiety(ETM), wherein the configuration of said detection electrode results inmixing of said sample; and b) detecting the presence of said ETM usingsaid detection electrode.
 28. A method according to claim 27 whereinsaid detection electrode comprises weirs.
 29. A method of detecting atarget analyte in a sample comprising: a) flowing said sample past adetection electrode comprising a covalently attached capture ligandunder conditions that result in the formation of an assay complexcomprising said target analyte and said capture ligand, wherein saidassay complex further comprises at least one electron transfer moiety(ETM), wherein said detection electrode is porous and is positioned suchthat said sample flows through said electrode; and b) detecting thepresence of said ETM using said detection electrode.